Highly sensitive detection method for cancer cell-derived extracellular endoplasmic reticulum genes utilizing fusion reaction with liposomes

Abstract

The present invention successfully introduced a new approach to target specific EV subpopulations based on charge-mediated fusion of EVs with CLIP. By adjusting the surface charge of liposomes through the ratio of positively and negatively charged lipids, we identified the optimal ratio for efficient and stable fusion with exosomes. Taking advantage of CLIP's high fusion rate, rapidity, and broad applicability, the present method demonstrated excellent sensitivity and selectivity for tumor-derived EV miRNAs in a lysis-free manner using droplet microfluidics. In particular, the EV-CLIP method enabled digital detection of EGFR L858R and T790M mutations without full sample processing, simplifying the detection process and preventing EV loss.

Description

[Technical Field]

[0001] The present invention relates to charged liposomes for detecting extracellular vesicles (EVs) and uses thereof, and more particularly to liposomes for detecting extracellular vesicles containing cationic lipids and neutral lipids, compositions for detecting cancer cell-derived extracellular vesicles containing the liposomes, detection kits, detection methods, cancer diagnostic compositions, cancer diagnostic kits, methods for providing information for cancer diagnosis, and cancer diagnostic methods. [Background technology]

[0002] Single extracellular vesicle (EV) analysis has emerged as a powerful tool in biomedical science, providing valuable insights into a variety of diseases and disorders (Sullivan, LB, Nature Chemical Biology 2017, 13(9), 924-925). Identifying unique nucleic acids (Cocks, A. et al., Seminars in Cancer Biology 2021, 75, 127-135), proteins (Whittle, K. et al., Critical Research in Oncology / Hematology 2022, 171, 103603), or other molecules that are indicative of this specific disease (Yu, W. et al., Ann Oncol 2021, 32(4), 466-477), detailed investigation of EV heterogeneity (Tkach, M.; Thery, C., Cell 2016, 164(6), 1226-1232), identification of disease-specific biomarkers (Peinado, H. et al., Nature Medicine 2012, 18(6), 883-891), and monitoring dynamic changes in disease progression will become possible.

[0003] Therefore, investigating the contents and properties of single EVs may reveal important information about disease processes (Marar, C., et al., Nature Immunology 2021, 22(5), 560-570). Standard quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is optimal for detecting EV-derived RNA with excellent sensitivity (Gandham, S., et al., Trends Biotechnol 2020, 38(10), 1066-1098), and various novel approaches have been proposed (Shao, H., et al., Chem REV 2018, 118(4), 1917-1950). Despite the remarkable advantages offered by these methods, most of these detection methods still present significant challenges, particularly the laborious and time-consuming steps involved in EV isolation from biological samples such as plasma, EV lysis, RNA extraction, and reverse transcription amplification (Erdbrugger, U.; Lannigan, J., Cytometry Part A 2016, 89(2), 123-134). Furthermore, biological samples often contain a mixture of tumor cell-derived EVs and non-tumor cell-derived EVs. While traditional RNA isolation and analysis methods involve bulk solutions, distinguishing between tumor cell-derived and non-tumor cell-derived EVs remains challenging (Bordanaba-Florit, G., et al., Nature Protocols 2021, 16(7), 3163-3185). Consequently, further research is essential to develop more efficient and accurate strategies for tumor cell-derived EV RNA detection.

[0004] Calcium ions (Ca) are often mediated by SNARE proteins. 2+Membrane fusion, promoted by lipid bilayers (PBLs), is essential for various cellular processes, including exocytosis, endocytosis, membrane remodeling, cell division, signal transduction, and intracellular trafficking (Koike, S.; Jahn, R., Nature Communications 2019, 10(1), 1608). Extensive research on the fusion mechanism of phospholipid compartments has been carried out (Ma, M.; Bong, D., Accounts of Chemical Research 2013, 46(12), 2988-2997), and has been applied to the development of functional systems applicable to diagnostics and therapy (Mazur, F.; Chandrawati, R., Chem Nano Mat 2021, 7(3), 223-237). Various EV membrane fusion processes have been proposed, such as pH-dependent (Yang, Y., et al., Advanced Materials 2017, 29(13), 1605604), polyethylene glycol-mediated (Piffoux, M., et al., ACS Nano 2018, 12(7), 6830-6842), catechol-metallosupramolecular complex (Kumar, S., et al., Nature Catalysis 2021, 4(9), 763-774), freeze-thaw cycle-mediated (Cheng, L., et al., Biomaterials 2021, 275, 120964), and DNA zipper-mediated (Peruzzi, J.A., et al., Angew Chem Int Ed Engl 2019, 58(51), 18683-18690). Various molecular components on the plasma membrane that are connected or docked to the membrane are associated, bringing them into close proximity and inducing localized turbulence, reducing the energy barrier for fusion.Recently, numerous strategies have been developed to detect EV RNA in its natural environment, utilizing fusogenic vesicles inspired by viral infection mechanisms (Gao, X., et al., Angewandte Chemie International Edition 2019, 58(26), 8719-8723) and aptamer-mediated fusion (Feng, J., et al., Analytical Chemistry 2023, 95(19), 7743-7752). However, these methods still require complex genetic manipulation and extensive aptamer optimization, which are technically challenging and time-consuming, limiting their usefulness in a wide range of clinical scenarios. Therefore, it is important to develop a more generalized and simplified approach for detecting tumor-derived EV RNA that can be easily applied clinically.

[0005] In this study, we successfully developed a simple and efficient charge-induced fusion method for EV / liposome fusion in a microfluidic droplet reactor, regardless of the type of surface protein, enabling investigation of miRNA and mRNA within individual EVs. By fine-tuning the surface charge of liposomes through manipulation of the ratio of positively and negatively charged lipids, we were able to enable efficient fusion with EVs, precisely controlling fusion rates from less than 5% to over 60%. A specific combination of charged liposomes demonstrated the highest fusion efficiency among different charge types. Furthermore, this study enables highly efficient profiling of miRNA or mRNA within single EVs by sorting individual EVs in emulsion droplets and utilizing charged-liposome (CLIP) EV detection (EV-CLIP) through droplet scanning. The inventors successfully digitally detected EGFR L858R and T790M mutations in plasma samples from 73 lung cancer patients (17 patients without mutations and 56 patients with mutations) and 10 healthy donors. In particular, the innovative EV-CLIP method simplified the detection process without full sample processing and minimized EV loss.

[0006] The above information provided in this Background Art section is merely intended to enhance understanding of the background of the present invention, and therefore may not include information that constitutes prior art already known to those skilled in the art to which the present invention pertains. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a liposome (charged-liposome) for detecting cancer cell-derived extracellular vesicle (EV) genes with high sensitivity and high selectivity.

[0008] Another object of the present invention is to provide a composition for detecting cancer cell-derived extracellular endoplasmic reticulum, a detection kit, a detection method, a cancer diagnostic composition, a cancer diagnostic kit, a method for providing information for cancer diagnosis, and a cancer diagnostic method, which contain the above-mentioned liposome. [Means for solving the problem]

[0009] To achieve the above object, the present invention provides a liposome for detecting cancer cell-derived extracellular vesicles (EVs), which comprises a cationic lipid and a neutral lipid, and is characterized by encapsulating a cancer cell-specific molecular beacon within the liposome.

[0010] The present invention also provides a composition and a kit for detecting cancer cell-derived extracellular endoplasmic reticulum, which contain the above-mentioned liposome for detecting extracellular endoplasmic reticulum.

[0011] The present invention also provides a method for detecting extracellular vesicles derived from cancer cells, which comprises the step of fusing the liposome for detecting extracellular vesicles with extracellular vesicles derived from a biological sample.

[0012] The present invention also provides a cancer diagnostic composition and a cancer diagnostic kit, each containing the above-mentioned liposome for detecting extracellular endoplasmic reticulum.

[0013] The present invention also provides a method for providing information for cancer diagnosis and a method for diagnosing cancer, which comprises fusing the liposome for detecting extracellular vesicles with extracellular vesicles derived from a biological sample. [Brief explanation of the drawings]

[0014] [Figure 1]Conceptual diagram of EV RNA analysis based on EV-CLIP fusion reactions. (a) Schematic diagram of the method for detecting specific gene mutations within EVs by inducing a fusion reaction between CLIP loaded with a molecular beacon (MB) tailored to the RNA to be analyzed and EVs in a droplet reactor. For example, to detect the EGFR L858R mutation, a gene associated with targeted therapeutic selection for lung cancer patients, MBs are tagged with green fluorescent molecules, while MBs associated with EGFR T790M, associated with drug resistance, are tagged with red fluorescent molecules. The MB reacts with the mutant gene within the EVs in each droplet reactor to emit a fluorescent signal. The number of droplets with detected fluorescent signals is counted and used for digital quantitative analysis of EV-derived RNA. (b) Schematic diagram of the CLIP (charged-liposome) formulation using the microfluidic hydrodynamic focusing method. CLIP is composed of positively charged 1,2-dioleoyl-3-trimethylammonium propane (DOTAP, red) and negatively charged 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, green) lipids. (c) Image of the CLIP surface taken with a transmission electron microscope (TEM) (diameter: 92.5 ± 5.65 nm, n = 3 with 9 data points). Scale bar: 100 nm. (d) Zeta potential of CLIP with various mole ratios (%) of DOTAP and DOPC (χ DOTAP :χ DOPC) (0% DOTAP: -18.63 ± 0.76, 25% DOTAP: 16.17 ± 3.05, 50% DOTAP: 26.90 ± 5.27, 75% DOTAP: 32.20 ± 2.26, 100% DOTAP: 38.67 ± 2.52). Data are shown as mean ± SD and are based on three independently synthesized CLIP configurations. (e) Hydrodynamic size (DH) distribution of CLIP with different DOTAP ratios. Size and surface charge measurements were performed using DLS (0% DOTAP: 95.82 ± 8.87 nm, 25% DOTAP: 116.40 ± 3.67 nm, 50% DOTAP: 113.40 ± 1.00 nm, 75% DOTAP: 115.03 ± 8.86 nm, 100% DOTAP: 113.7 ± 4.3 nm). Data represent the mean ± SD and are based on three independently synthesized CLIP configurations.All statistical analyses were performed with one-way ANOVA. [Figure 2] Experimental setup for liposome generation based on microfluidic hydrodynamic focusing (MHF). The setup consists of a digital microscope, tip stand, oily and aqueous phase pumps, a computer, and a five-input chip. Lipids dissolved in ethanol (EtOH) are loaded into the oily phase pump, and PBS is loaded into the aqueous phase pump. The oily phase pump pushes lipids into the left side of the chip, and the aqueous phase pump pushes PBS into the right side of the chip. Through appropriate flow control, the oily phase is sandwiched between the two aqueous phases, forming liposomes inside the five-input chip. [Figure 3] Liposome size distribution measured by nanoparticle tracking analysis (NTA). Liposomes exhibit a single-peak distribution curve, indicating a homogeneous liposome population. [Figure 4] Stability of various CLIPs over time. Liposome size was measured over time to assess changes in size over a 48-hour period. The sizes of 0%, 25%, 50%, and 75% DOTAP liposomes remained stable over time, while the size of 100% DOTAP liposomes increased steadily to 300 nm after 48 hours (6 hours: 160.53 ± 8.15 nm, 12 hours: 188.27 ± 7.58 nm, 24 hours: 230.37 ± 14.96 nm, 48 hours: 311.50 ± 8.51 nm). [Figure 5] EV isolation. (a) ExoDisc platform (ExoDisc, LabSpinner), a centrifugal disk equipped with a bipolar aluminum filter with a pore diameter of 0.02 μm. The supernatant was passed through the filter by centrifugation at 3000 rpm (approximately 500 g), and 100 μL of concentrated EVs were collected in the collection chamber and resuspended in 1x PBS at a dilution factor of 2. (b) Tabletop centrifuge platform for ExoDisc operation. (c) ExoDisc operating principle. (d) EVs were characterized by nanoparticle tracking analysis (NTA). [Figure 6]Droplet generation experimental setup. The setup consisted of a digital microscope, tip stand, oil pump, two sample pumps, a sample reservoir, a droplet outlet, a droplet chip, a computer, and a droplet reservoir chip. EVs and CLIPs were loaded separately into the two sample pumps, and FC-40 surfactant was loaded into the oil pump and sample reservoir. The oil pump pushed FC-40 into the vertical side of the T-junction, and the sample pump pushed EVs and CLIPs into the sample channel. The EVs and CLIPs met at the T-junction to form water-in-oil droplets, which were collected at the droplet outlet. The droplets were then imaged using the droplet reservoir chip under a fluorescence microscope for analysis. [Figure 7]Controlled fusion of EVs and CLIP. (a) Schematic of CLIP and EV fusion using a droplet generation chip. The microfluidic device was designed to generate a water-in-oil droplet reactor at a flow-focusing junction. Two aqueous phases (EVs and CLIP) were delivered to an oil stream (FC-40 surfactant PFPE-PEG) to meet and form droplets. (b) Schematic of Förster Resonance Energy Transfer (FRET)-based lipid mixing assay to analyze the EV and CLIP fusion reaction. The average distance between the FRET pair donor (green, nitrobenzoxadiazole) and acceptor (red, rhodamine) lipid probes increases and the FRET efficiency decreases following charge-based fusion of labeled CLIP membranes and unlabeled EV membranes (PE: phosphoethanolamine, PS: phosphatidylserine). (c) Analysis of the degree of lipid mixing in fused vesicles as a function of the EV and CLIP fusion ratio and the DOTAP percentage in the CLIP composition. All statistical analyses were performed by one-way analysis of variance using Dunnett's multiple comparison test. (d) Confocal laser scanning microscopy (CLSM) images of EVs, CLIP, and fused vesicles, showing the colocalization of EVs (green) and CLIP (red). Confocal laser scanning microscopy (CLSM) images of high-resolution images of EVs, CLIP, and fused vesicles (EV-CLIP) at three ratios of EV to CLIP (75% DOTAP) (Mall ratios of 10:1, 1:1, and 1:10). Intensity analysis shows the colocalization of EVs (green) and CLIP (red). (e) Graph showing the proportion of each vesicle in the analysis results in (d). The number of vesicles ranged from 100 to 160, analyzed from 10 independent images. Statistical analysis was performed by one-way analysis of variance. (f) Representative images of CLIP, EVs, and fused vesicles taken with a transmission electron microscope (TEM) (fused vesicle diameter: 174.85 ± 13.83 nm, n = 5). Scale bar: 100 nm. (g) Controlled vesicle size before and after fusion (EVs: 137.00 ± 14.30 nm, CLIP: 115.03 ± 8.86 nm, EVs-CLIP: 174.10 ± 10.28 nm). Data are shown as mean ± SD, n = 3.All statistical analyses were performed by one-way analysis of variance using Tukey's multiple comparison test. (h) Zeta potential of endoplasmic reticulum before and after control fusion. Data are shown as mean ± SD, n = 3. All statistical analyses were performed by one-way analysis of variance using Tukey's multiple comparison test. [Figure 8] The droplet size distribution graph was measured using the Droplet Monitor application, and the average droplet size was confirmed to be 27.40 ± 2.06 μm. [Figure 9] Increase in size and zeta potential of fused vesicles as a function of DOTAP percentage. (a) Graph showing the increase in size of fused vesicles as a function of DOTAP percentage (0% DOTAP: 135.9 ± 24.7 nm, 25% DOTAP: 150.5 ± 24.3 nm, 50% DOTAP: 161.6 ± 11.9 nm, 75% DOTAP: 174.1 ± 10.3 nm, 100% DOTAP: 231.3 ± 42.8 nm). Each data point represents the mean ± SD, n = 3. All statistical analyses were performed using one-way ANOVA. (b) Graph showing the change in surface charge of fused vesicles as a function of DOTAP percentage (0% DOTAP: -18.4 ± 6.9, 25% DOTAP: -12.3 ± 3.0, 50% DOTAP: -9.24 ± 4.55, 75% DOTAP: -3.74 ± 3.80, 100% DOTAP: -0.71 ± 4.39). Each data point represents the mean ± SD, n = 3. All statistical analyses were performed using one-way ANOVA. [Figure 10] Pearson correlation coefficient (PCC) values ​​for fused vesicles, liposomes, and EVs. Pearson correlation coefficients (PCCs) were measured for 10 different single vesicles and calculated using the JaCoP algorithm in ImageJ. The PCCs for fused vesicles were 0.690 ± 0.096, for liposomes -0.0139 ± 0.01264, and for EVs 0.0021 ± 0.0066. [Figure 11]Effect of Rho-NBD addition to liposomes on zeta potential. Zeta potential changes of liposomes with various DOTAP ratios (0%, 25%, 75%, 100% DOTAP). Each data point represents the mean ± SD, n=3. All statistical analyses were performed using two-tailed unpaired Student's t-test. [Figure 12] TEM images and size comparison of aggregates formed during bulk-scale EV-liposome fusion. (a) TEM image showing aggregates formed when EV-liposome fusion is performed at a bulk scale. Scale bar: 100 nm. (b) Size comparison of fused vesicles in droplets and bulk scales. Average size of fused vesicles in droplets: 174.1 ± 10.28 nm; average size of fused vesicles in bulk scale: 1173.17 ± 508.58 nm. Each data point represents the mean ± SD, n = 3. All statistical analyses were performed using a two-tailed unpaired Student's t-test. [Figure 13] Effect of MB insertion into liposomes on size and zeta potential. (a) A graph showing the size distribution of three different liposome populations (without molecular beacon (MB); with miR-21-detecting MB; with EGFR mutation-detecting MB). (b) A graph showing the zeta potential of three different liposome populations (without molecular beacon (MB); with miR-21-detecting MB; with EGFR mutation-detecting MB). Each data point represents the mean ± SD, n = 3. All statistical analyses were performed using one-way ANOVA. [Figure 14]Molecular beacon-based tumor EV detection. (a) Schematic diagram showing CLIP production using an MHF chip, fusion reaction with EVs using a droplet reactor, and detection process using a detection chamber chip. (b) Photograph of the above three chips. (c) Representative fluorescence images of molecular beacon-based detection of miR-21 in H1975 cell-derived EVs at various concentrations in phosphate-buffered saline (PBS). (d) Concentration-related miR-21 detection results for H1975 cell-derived EVs. Data are shown as mean ± SD, n = 3. (e) The limit of detection (LOD) was confirmed at 79 EVs / μL. (f) Graph showing the results of molecular beacon-based detection of miR-21 in normal subjects and cancer patients, confirming the existence of differences. [Figure 15] Molecular beacon-based tumor EV detection. (a) Representative fluorescence images of molecular beacon-based detection of L858R, T790M, and L858R+T790M mutations in H1975 cell-derived EVs in phosphate-buffered saline (PBS). (b) Concentration-related detection of L858R and T790M mutations in H1975 cell-derived EVs in phosphate-buffered saline (PBS). Data are shown as mean ± SD, n = 3. The limits of detection (LOD) were 1348 EVs / μL and 3595 EVs / μL, respectively. (c) Concentration-related detection of L858R and T790M mutations in H1975 cell-derived EVs in human plasma. Data are shown as mean ± SD, n = 3. The limits of detection (LOD) were 1348 EVs / μL and 2926 EVs / μL, respectively. (d) Results of droplet digital polymerase chain reaction (ddPCR) performed after EV isolation. Each data point represents the mean ± SD, n = 3. (e) Correlation of detection readings for EGFRL858R and T790M mutations in PBS and human plasma. Data represent the mean ± SD of independent analyses, n = 3. [Figure 16] Droplet Image Processing Flowchart: The flow chart describes the steps in processing droplet images for analysis, from image acquisition to processing, quantification and analysis. [Figure 17]Detection of EGFR L858R and T790M mutations using the fusion system in lung cancer patient samples. (A) Schematic diagram of EGFR L858R and T790M mutation detection in 10 healthy donors and 73 lung cancer patients using EV-CLIP fusion from normal and patient plasma samples. (B) Droplet detection percentages for L858R and T790M mutations in 10 normal donors, 17 lung cancer patients without EGFR mutations, and 56 lung cancer patients with EGFR mutations. (c) Comparison of L858R and T790M mutation detection by group. (d) Receiver Operating Characteristic (ROC) curves for L858R and T790M mutation detection. The AUC (area under the curve) was 1.0 for L858R and 0.9762 for T790M. Each data point represents the average of three independent replicates. All statistical analyses were performed using one-way ANOVA. DETAILED DESCRIPTION OF THE INVENTION

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0016] Investigating the protein-free fusion mechanism between individual extracellular vesicles (EVs) and liposomes not only improves our understanding of diverse EV populations but also allows us to investigate the RNA content of single EVs, which can identify distinct subpopulations in disease states. However, such analytical techniques require EV lysis, a costly, labor-intensive, and time-consuming process that often results in reduced sensitivity. We developed an approach for detecting distinct EV subpopulations based on charge-mediated fusion of EVs and liposomes. The surface charge of liposomes was tuned by varying the ratio of positively and negatively charged lipids, and specific ratios demonstrated highly efficient and stable fusion with exosomes without damaging membrane properties, as confirmed by membrane-mixing assays. Taking advantage of the high fusion rate, rapid applicability, and wide range of potential applications, charged-liposome EV detection (EV-CLIP) demonstrated remarkable sensitivity and selectivity for EV-derived RNA in a lysis-free manner using droplet microfluidics. Furthermore, EGFR L858R and T790M mutations were digitally detected through EV-CLIP in plasma samples collected from 73 lung cancer patients (17 mutation-free and 56 mutation-positive) and 10 healthy donors, without a prior EV isolation step, further simplifying the detection process and preventing EV loss. Overall, EV-CLIP holds great potential in clinical settings for the accurate quantification of rare EV subpopulations, providing new opportunities to explore fundamental questions in cancer biology.

[0017] Therefore, in one aspect, the present invention relates to a liposome for detecting cancer cell-derived extracellular vesicles (EVs), which contains a cationic lipid and a neutral lipid, and is characterized in that a cancer cell-specific molecular beacon is encapsulated within the liposome.

[0018] As used herein, "extracellular vesicles (EVs)" are small secretory cells (generally about 30-800 nm) that can contain nucleic acids, proteins, or other biomolecules, including exosomes and microvesicles. EVs can act as cellular messengers by transporting biomolecular materials to various locations in an organism or biological system.

[0019] In the present invention, the "cancer cell-specific molecular beacon" refers to a molecular beacon that specifically binds to a cancer-specific substance that is not present in extracellular endoplasmic reticulum derived from normal cells but is present in extracellular endoplasmic reticulum derived from cancer cells.

[0020] In the present invention, the cancer-specific substances present in the cancer cell-derived extracellular vesicles include microRNA-21 (miR-21), microRNA-25 (miR-25), microRNA-27 (miR-27), microRNA-54 (miR-54), microRNA-155 (miR-155), microRNA-210 (miR-210), microRNA-375 (miR-375), microRNA-451 (miR-451), microRNA-486 (miR-486), microRNA-495 (miR-495), microRNA-574-3p (miR-574-3p), EGF L858R mutation, EGFR T790M mutation, GPC1, KRAS, AR-V7, survivin, TK-1, c-Myc, GalNAc-T, Cyclin D1, etc. (Wei Pan, et al. al., Anal.Chem.2013, 85, 21, 10581-10588; Xiang-Hong Peng, et al., Cancer Res(2005)65(5):1909-1917), but are not limited thereto.

[0021] As used herein, "molecular beacon (MB)" refers to an oligonucleotide that forms a hairpin-shaped secondary structure and is tagged at its 3' end with a quencher substance. During annealing, the molecular beacon probe specifically hybridizes to the template gene at a complementary region, increasing the distance between the fluorescent substance and the quencher substance, thereby releasing the inhibition of luminescence by the quencher substance and emitting fluorescence. On the other hand, unhybridized molecular beacons maintain their secondary structure and do not emit fluorescence due to inhibition by the quencher.

[0022] In one embodiment of the present invention, molecular beacons represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, were used to detect EGFRL858R and T790M mutations in the H1975 lung cancer cell line, and tumor-derived extracellular vesicles (tEVs) were detected using a molecular beacon represented by the nucleotide sequence of SEQ ID NO: 3, which was designed to target microRNA-21 (or miR-21), which is known to be upregulated in various tumor types such as breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, and gastric cancer.

[0023] As used herein, the term "lipid" or "lipid-like substance" refers to a molecule containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are generally poorly soluble in water. In aqueous environments, the amphiphilic nature of the molecules allows them to self-assemble into organized structures and other phases. One of these phases is comprised of a lipid bilayer when they exist in aqueous environments as vesicles, multilamellar / unilamellar liposomes, or membranes. Hydrophobic groups include nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups, such as carbohydrates, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups.

[0024] As used herein, the term "amphiphilic" refers to a molecule that has both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. The polar portion can also have a formal positive or negative charge. Alternatively, the polar portion can have both a formal positive and negative charge and can be a zwitterion or inner salt. For purposes of this specification, an amphiphilic compound can be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0025] The terms "lipid-analog," "lipid-analog compound," or "lipid-analog molecule" refer to substances that are structurally and / or functionally related to lipids but may not be considered lipids in the strict sense. For example, this term includes surfactants or synthetic compounds that have both hydrophilic and hydrophobic portions, including compounds that can form amphiphilic layers when present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Generally speaking, this term refers to molecules that contain hydrophilic and hydrophobic portions with different structural organizations that may or may not resemble those of lipids. In this application, the term "lipid" should be interpreted to encompass both lipids and lipid-analogs, unless the context clearly indicates otherwise.

[0026] As used herein, "cationic lipid" or "cationic lipid-analog" refers to a lipid or lipid-analog that has a net charge. Cationic lipids or lipid-analogs bind to negatively charged nucleic acids through electrostatic interactions. Cationic lipids generally have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl, or larger acyl chain, and the lipid head group generally carries a positive charge.

[0027] In certain embodiments, the cationic lipid or lipid-like substance has a net charge only at certain pHs, particularly acidic pHs, while preferably lacking any net charge at higher pHs, such as physiological pH, and is uncharged, i.e., neutral. Such ionizable behavior is believed to aid endosomal escape, reduce toxicity, and improve efficacy compared to particles that remain cationic at physiological pH.

[0028] In the present invention, the cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-dimethylamino propane (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-chol), dimethyldioctadecylammonium bromide (DODAB), 1,2-dioleoyl-3-dimethylammonium propane ... propane (DODAP), dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dimyristyloxy-propyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine), 1,2-dimyristoyl-3-trimethylammonium propane (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine), 1,2-dimyristoyl-3-trimethylammonium propane (1,2-dimyristoyl-3-trimethylammonium propane).DMTAP), 1,2-Dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIA), 2,3-Dioleyoxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadiene), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadiene), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxape (3-dimethylamino-2-(cholest-5-en-3-β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane; CLinDMA), 2-[5'-(cholest-5-en-3β-oxy)-3'-oxape ntoxy]-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane; CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (N,N-dimethyl-3,4-Dioleyloxybenzylamine; DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane;DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-KDMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (2,2-dilinoleyl-4-d 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (heptatri aconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate; DLin-MC3-DMA), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide;GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide ((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide; GAP-DMRIE), (±)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP), and 1,2-dipalmitoyl-3-dimethylammonium propane. (1,2-dipalmitoyl-3-dimethylammonium-propane; DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide;MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocoline (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide) bromide (DMORIE), Di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioateL319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino-ethyl)-amino]-ethylamino)propionamide; lipid 98N12-5) and 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]et;

[0033] The present invention can be characterized in that the hydroxybenzoate is selected from the group consisting of, but not limited to, [hydroxybenzoate] ...

[0029] In the present invention, the liposomes can contain one or more anionic lipids and / or non-cationic lipids, such as neutral lipids.

[0030] As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of a number of lipid species that either exhibit no charge or exist in a neutral zwitterionic form at a selected pH.

[0031] In the present invention, the neutral lipids include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine ... The phospholipid may be characterized by being selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PD), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA), and phosphatidylcholine (PC), but is not limited thereto.

[0032] Preferably, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), but the present invention is not limited thereto.

[0033] In the present invention, the molar ratio (%) of the cationic lipid in the liposome can be characterized as being 25 to 75%, but is not limited thereto.

[0034] In one embodiment of the present invention, when 25% DOTAP was included in the total lipids, the surface charge shifted significantly from negative to positive, and the surface charge remained positive up to 100% (Figure 1d), increasing the fusion rate with extracellular vesicles (EXP) (Figure 7c). However, despite the highest fusion efficiency, CLIP containing 100% DOTAP exhibited instability over time and formed aggregates (Figures 4 and 12). Therefore, the molar ratio (%) of cationic lipids in the liposomes is preferably 75%, and the molar ratio of cationic lipids to neutral lipids is preferably 75:25, but this is not intended to be limiting.

[0035] In another aspect, the present invention relates to a composition for detecting cancer cell-derived extracellular endoplasmic reticulum, which comprises the above-mentioned liposome for detecting extracellular endoplasmic reticulum.

[0036] In still another aspect, the present invention relates to a kit for detecting cancer cell-derived extracellular endoplasmic reticulum, which comprises the above-mentioned liposome for detecting extracellular endoplasmic reticulum.

[0037] In another aspect, the present invention relates to a method for detecting extracellular vesicles derived from cancer cells, which comprises the steps of fusing the liposome for detecting extracellular vesicles with extracellular vesicles derived from a biological sample; and determining that the extracellular vesicles are derived from cancer cells when a fluorescence signal is generated.

[0038] In the present invention, the fusion may be characterized as being performed in a droplet reactor, but is not limited thereto.

[0039] In the present invention, the droplet reactor may be characterized by comprising two aqueous phase channels, one oil phase channel, one junction, and one outlet channel, and may be characterized by introducing liposomes and extracellular vesicles derived from a biological sample into the two aqueous phase channels, respectively, to generate aqueous droplets at the junction, but is not limited thereto.

[0040] In one embodiment of the present invention, to ensure precise control over the stoichiometry of EVs (EVs and CLIPs) and prevent undesired aggregation, the entire fusion process was carried out in a droplet reactor using a precisely controllable μencapsulator, minimizing potential aggregation that can occur in bulk-scale reactions (Figure 6). The μencapsulator operated by flowing EVs and CLIPs in separate phases through two aqueous inlets at a flow rate of 1.5 μL / min, while a continuous oily phase inlet delivered the biosynthetic surfactant FC-40 at a flow rate of 35 μL / min, enabling the formation of aqueous droplets containing two EVs when they met at the junction in the oily phase (Figure 7a).

[0041] In the present invention, the molar ratio of the liposome to the extracellular vesicles derived from the biological sample may be characterized as being 1:1 to 10:1, but is not limited thereto.

[0042] In one embodiment of the present invention, the lipid membrane mixing percentage was 35.67±1.35% when the molar ratio of extracellular vesicles to liposomes was 10:1, 45.91±6.33% when the molar ratio was 1:1, and 71.45±6.58% when the molar ratio was 1:10. Therefore, the molar ratio of extracellular vesicles to liposomes is preferably 1:10, but is not limited thereto.

[0043] In one embodiment of the present invention, EGFR L858R and T790M mutations can be detected digitally in plasma samples collected from lung cancer patients and healthy donors using charged-liposome EV detection (EV-CLIP) without a prior EV isolation step, and it was confirmed that the simplified detection process minimizes EV loss.In other words, by fusing sample-derived EVs with the liposomes of the present invention and confirming whether a fluorescent signal is generated, it is possible to easily determine whether a sample contains cancer cells.

[0044] As used herein, the term "biological sample" includes biological-fluid samples and biological tissue samples.

[0045] As used herein, the term "biological fluid" refers to any fluid isolated or derived from an organism, including prokaryotes, eukaryotes, bacteria, fungi, yeast, invertebrates, vertebrates, reptiles, fish, insects, plants, and animals, and may include, but is not limited to, serum, plasma, whole blood, urine, saliva, breast milk, tears, sweat, synovial fluid, cerebrospinal fluid, semen, vaginal fluid, sputum, pleural fluid, lymphatic fluid, peritoneal fluid, and amniotic fluid. Bronchial lavage fluid and culture fluid collected from cultured cells (e.g., cell culture supernatant, conditioned culture fluid, cell culture medium, or cell culture medium) may also be biological fluids.

[0046] As used herein, the term "biological tissue" refers to a population of cells derived from prokaryotes, eukaryotes, bacteria, fungi, yeast, invertebrates, vertebrates, reptiles, fish, insects, plants, or animals. It can also be cultured biological tissue. Non-limiting examples of biological tissue samples include surgical samples, biopsy samples, tissue, feces, plant tissue, insect tissue, and cultured cells.

[0047] In another aspect, the present invention relates to a composition for diagnosing cancer, which comprises the liposome for detecting extracellular endoplasmic reticulum.

[0048] In still another aspect, the present invention relates to a cancer diagnostic kit comprising the above-mentioned liposome for detecting extracellular endoplasmic reticulum.

[0049] In another aspect, the present invention relates to a method for providing information for cancer diagnosis and a method for diagnosing cancer, which comprises the steps of fusing the liposome for detecting extracellular vesicles with extracellular vesicles derived from a biological sample; and determining that the cancer is present when a fluorescence signal is generated.

[0050] The present invention may be characterized in that it is carried out in a droplet reactor including two aqueous phase channels, one oil phase channel, one junction, and one outlet channel, but is not limited thereto.

[0051] In one embodiment of the present invention, EGFR L858R and T790M mutations can be detected digitally in plasma samples collected from lung cancer patients and healthy donors using charged liposome EV detection (EV-CLIP) without a prior EV isolation step. Specifically, by fusing sample-derived EVs with the liposomes of the present invention and confirming whether a sample contains cancer, it is possible to easily determine whether the sample contains cancer. High levels of mutations can be detected in a short time without any processing, even in early-stage cancers, allowing for easy differentiation between cancer patients and healthy donors. This demonstrates the value of this method over conventional methods in converting tumor-derived EV miRNAs into standard clinical indicators for cancer diagnosis.

[0052] In the present invention, the cancer may be characterized by being selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung cancer, peritoneal cancer, colon cancer, biliary tract tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, thyroid cancer, oral cancer, osteosarcoma, gallbladder cancer, biliary tract cancer, kidney cancer, bladder cancer, renal cell carcinoma, melanoma, brain cancer, glioma, glioblastoma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, small intestine cancer, esophageal cancer, colon cancer, gastric cancer, urethral cancer, cervical cancer, prostate cancer, ovarian cancer, metastatic cancer, head and neck cancer, rectal cancer, non-Hodgkin's lymphoma, multiple myeloma, acute myeloid leukemia, lymphoma, acute lymphoblastic leukemia, and chronic myeloid leukemia, but is not limited thereto.

[0053] In the present invention, the diagnostic kit may additionally contain various components, such as one or more other component compositions, solutions, or devices suitable for the diagnostic method. The diagnostic kit may additionally contain a compartmented carrier means for containing a biological sample, a container containing a reagent, etc. The carrier means is suitable for containing one or more containers, such as a bottle or tube, each containing an independent component used in the method of the present invention. From this specification, one skilled in the art can easily dispense the necessary formulations into the containers. [Example]

[0054] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0055] Example 1. Materials and Methods Example 1-1: Reagents and equipment 0.2 μm syringe filter (Minisart® NML Syringe Filters, S6534), 1× phosphate buffered saline (pH 7.4, Gibco), antibiotic / antimycotic (Gibco, 15240062), anti-CD63 (BD ​​Bioscience, BD556019), anti-CD81 (BD Bioscience, BD555675), anti-CD9 (BD Bioscience, BD55370), anti-EGFR (abcam, ab192263), EV-depleted FBS (Systems Biosciences Inc), ExodiscTM-C (LabSpinner), molecular beacon (Oligo, Macrogen), NBD-PE (N-(7-nitrobenz-2-o×a-1, 3-diazol-4-yl)-1,2-dihe×adecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt, Thermofisher, N360), Rhodamine-DHPE (Lissamine rhodamine B1,2 dihe×adecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt, Thermofisher, L1392), TMB solution (Sigma-Aldrich, T0440-100ML).

[0056] 0.2 μm syringe filter (Minisart® CA Syringe Filters, 16534K), μencapsulator 2 reagent droplet chip (Dolomite Microfluidics 3200529), 100K Amicon centrifugal filter (Merck milipore, UFC810096), 150 μm aqueous 5-input chip 3D (Dolomite Microfluidic 3200834), 30 μ chamber chip (Microfluidic ChipShop, 10001447), 96-well plate (Corning, 3364), confocal laser scanning microscope (Zeiss LSM 780NLO, Zeiss), Malvern Zetasizer (Nano ZS), NanoSight instrument (NanoSight NS500, Malvern Instruments), TECAN M2000 Pro Plate Reader (TECAN), transmission electron microscope (JEOL, JEM-2100).

[0057] Example 1-2: Cell culture H1975 cells (ATCC) were cultured in Dulbecco's medium supplemented with 5% (V / V) FBS (Gibco) and 1% Antibiotic / Antimycotic (100 U / mL penicillin and 100 mg / mL streptomycin, Gibco, 15240062). , s The cells were cultured in Modified Eagle Medium (DMEM) (Gibco, 11965092) under static conditions at 37°C in the presence of 5% CO2.

[0058] Example 1-3: Isolation of extracellular endoplasmic reticulum from cell culture medium Extracellular vesicles (EVs) were isolated from cell culture medium using a standardized Exo-Disc platform (Woo, H.-K., et al., AcsNano, 2017.11(2):pp.1360-1370). 70-80% confluent 10 cm culture dishes were washed twice with 1x phosphate-buffered saline (pH 7.4, Gibco) and cultured for 48 hours in medium supplemented with 5% EV-depleted FBS (Systems Biosciences Inc.) and 1% Antibiotic / Antimycotic. The cell culture supernatant was collected and centrifuged at 300g for 10 minutes, followed by another centrifugation at 2000g for 10 minutes to remove cell debris, and then passed through a 0.2μ syringe filter (Minisart® NML Syringe Filters, S6534). The purified supernatant was then concentrated using an ExoDisc platform (ExoDisc™-C, LabSpinner), a centrifugal disk equipped with a 0.02 μm pore diameter anodized aluminum filter. The supernatant was centrifuged at 3,000 rpm (approximately 500 g) to pass through the filter, and 100 μL of the concentrated EVs in the collection chamber were resuspended in 1x PBS at a dilution factor of 2. The isolated EVs were then analyzed using a NanoSight instrument and a Malvern Zetasizer to confirm their concentration, size, and zeta potential distribution. The EVs were then aliquoted and stored at -80°C until further use in experiments.

[0059] Examples 1-4: Liposome synthesis Liposomes were synthesized using a microfluidic hydrodynamic focusing method using a standardized platform (Dolomite Microfluidics). Lipid mixtures for liposomes were prepared by dissolving various ratios of 18:1 TAP or DOTAP (1,2-Dioleoyl-3-trimethylammonium propane, Avanti 890890P) and 18:1 (Δ9-Cis)PC or DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine, Avanti 850375P)—0%, 25%, 50%, 75%, and 100% DOTAP—in 1 mL of pure ethanol (Duksan Pure Chemicals, UN1170) filtered through a 0.2 μm syringe filter (Minisart® CA Syringe Filters, 16534K) to a final concentration of 5 mg / mL. CLIP synthesis was performed using a 150 μm hydrophilic 5-input chip 3D (Dolomite Microfluidic 3200834) with a flow rate of 50 μL / min for the aqueous phase (1× Phosphate Buffered Saline, pH 7.4, Gibco) and 5 μL / min for the oily phase (lipid mixture). The suspension was collected in a 1.5 mL microcentrifuge tube for 15 min, purified using a 100K Amicon centrifugal filter (Merck Milipore, UFC810096), washed twice with PBS, and analyzed using a NanoSight instrument (NanoSight MS500, Malvern Instruments) and a Malvern Zetasizer (Nano ZS) to measure concentration, size, and zeta potential distribution. The CLIP was then aliquoted and stored at 4°C until further use.

[0060] Examples 1-5: Microfluidic-Aided EV-CLIP Fusion Microfluidic-assisted fusion was performed using a commercial platform (Dolomite Microfluidics). The glass chip (μencapsulator 2 Reagent Droplet Chip, Dolomite Microfluidics 3200529) consisted of four channels (two aqueous phase channels, one oil phase channel, and one output channel). Liposomes and extracellular vesicles at the same concentration in PBS were loaded into the aqueous channels of the microfluidic chip, and droplets with a diameter of 30 μm were generated in the oil phase of FC-40 (RAN Biosciences, 008-FluoroSurfactant-2wtF-50G). The number of extracellular vesicles and liposomes was adjusted to an initial sample input concentration of 10–10. 5 The pump pressure was maintained at 2500 Bar and 1100 mBar for the oil and aqueous phases, respectively, and was adjustable to vary the particles / µL range. Droplets were collected in microcentrifuge tubes for 20 min and stored at 4°C until further use.

[0061] Examples 1-6: Detection of extracellular endoplasmic reticulum through fusion with MB-CLIP Extracellular vesicles detection was performed via the microfluidic-assisted fusion method described above. Various concentrations of extracellular vesicles (10–10), all in 1× PBS, were added. 5 A fixed concentration of molecular beacons, including 1000 particles / μL and CLIP (miR-21 or EGFR L858R and T790M mutations), was loaded into the aqueous channels of a microfluidic chip, generating 30 μm diameter droplets in an FC-40 oil phase. Pump pressures were maintained at 2500 mBar and 1100 mBar for the oil and aqueous phases, respectively. The droplet solution was collected in 1.5 mL amber microcentrifuge tubes for 20 min and stored at 4°C until further use in experiments.

[0062] Examples 1-7: EV-CLIP colocalization experiments For EV-liposome colocalization studies, EVs and liposomes were labeled with 3,3'-Dioctadecyloxacarbocyanine perchlorate (DiO) dye (Thermofisher Scientific, V22886) and Rhodamine-DHPE (Lissamine rhodamine B1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) (Thermofisher, L1392), respectively. For EV labeling, 100 μL of EVs pre-enriched on the ExoDisc platform were incubated with 2 μL of 200 nM DiO dye solution on a shaker at room temperature for 20 minutes. After incubation, the EVs were centrifuged at 3000 rpm and washed twice with 1x PBS. EVs enriched in the chamber were resuspended in 1x PBS at a dilution factor of 2. For liposome labeling, 1 mol% Rhodamine-DHPE was mixed into the lipid mixture. The volume was then adjusted to reach a final lipid concentration of 5 mg / mL. Rho-liposomes were synthesized using the microfluidic hydrodynamic focusing method described above. The synthesized liposomes were purified using a 100K Amicon centrifugal filter and washed with PBS to remove free dye. The dye-modified liposomes were then fused with extracellular vesicles using the microfluidic-assisted fusion method described above. The fused vesicles were then diluted with 1x PBS and imaged with a confocal laser scanning microscope (Zeiss LSM 780NLO) using a 1x10 Plan-Apochromat and a LU-NV laser unit with a 0.45NA objective lens equipped with 488 nm (green / Alexa Fluor 488) and 560 nm (red / Cy5). Colocalization analysis was performed using FIJI.

[0063] Examples 1-8: NBD-PE fusion quantitative analysis Fusion quantification was performed using a published method (Murtas, G., Systems and Synthetic Biology, 2010.4(2):pp.85-93). 1 mol% Rhodamine-DHPE (Lissamine rhodamine B 1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) (Thermofisher, L1392) and NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) (Thermofisher, N360) were mixed into the lipid mixture. The volume was then adjusted to reach a final lipid concentration of 5 mg / mL. Rho-NBD liposomes were synthesized using the microfluidic hydrodynamic focusing method described above. The synthesized liposomes were purified using a 100K Amicon centrifugal filter and washed with PBS to remove free dye. The dye-modified liposomes were fused with extracellular vesicles using the microfluidic-assisted fusion method described above. The fluorescent signal from the fused vesicles was measured using a spectrophotometer (M2000 Pro, TECAN) with excitation and emission wavelengths of 485 / 535 nm, respectively. The fusion percentage was calculated using the following formula:

number

[0064] Example 1-9: Molecular Beacon loading into liposome b miR-21-, EGFR L858R-, and EGFR T790M-targeting molecular beacons (Oligo, Macrogen) were loaded into liposomes by combining a fixed amount of molecular beacon in 1x PBS to a total concentration of 1 μM. Liposomes were then synthesized using the microfluidic hydrodynamic focusing (MHF) method with a final lipid concentration of 5 mg / mL. The miR-21-targeting beacon was loaded into one liposome compartment, while the EGFR L858R- and T790M mutation-specific beacons were simultaneously loaded into the other liposome compartment. The pump flow rates were maintained at 50 μL / min for the aqueous and 5 μL / min for the oily phases, respectively. The molecular beacon-loaded liposomes were then purified using a 100K Amicon centrifugal filter with two PBS washes and analyzed using a NanoSight instrument and a Malvern Zetasizer to confirm the concentration, size, and zeta potential distribution. The liposomes were then stored at 4°C before further use.

[0065] Examples 1-10: Droplet Imaging For imaging, 6 μL of droplet solution was loaded onto a 30 μm chamber chip (Microfluidic ChipShop, 10001447). Droplets were observed with a confocal laser scanning microscope (Zeiss LSM780NLO) using a 0.45 NA objective lens equipped with a ×10 Plan-Apochromat and an LU-NV laser unit at 488 nm (green / Alexa Fluor 488) and 560 nm (red / Cy5). All images for droplet analysis were processed using FIJI.

[0066] Examples 1-11: Healthy plasma spiking experiments Institutional Review Board (IRB)-approved healthy human plasma was obtained from the Red Cross (UNISTIRB-19-41-C). Human plasma was diluted in 1x PBS at a dilution factor of 10, followed by 10–10 5H1975 cell-derived EVs were spiked at various concentrations up to 1000 particles / μL. The spiked plasma samples were loaded onto droplet chips for microfluidic-assisted fusion of molecular beacon-loaded CLIP (miR-21 or EGFR L858R and T790M mutations). Pump pressures were maintained at 2500 mBar and 1100 mBar for the oil and aqueous phases, respectively. Droplets were collected in microcentrifuge tubes for 20 min and stored at 4°C until further analysis.

[0067] Examples 1-12: EGFR mutation detection in patient samples A total of 83 samples were obtained from 10 healthy donors, 17 lung cancer samples without mutations (6 stage I, 3 stage II, 3 stage III, and 5 stage IV), and 56 lung cancer samples with EGFR mutations (18 stage I, 6 stage II, 7 stage III, and 25 stage IV) at Inha University Hospital (2019-11-017), Chonnam National University Hwasun Hospital (CNUHH-2022-021), and Pusan ​​National University Hospital (2106-044-104). All healthy donor plasma samples were provided by Inha University Hospital, one stage II and one stage III sample without mutations, and 12 stage I, 5 stage II, 6 stage III, and 16 stage IV samples with EGFR mutations were provided by Chonnam National University Hwasun Hospital, and the remaining samples were provided by Pusan ​​National University Hospital. 20 μL of patient sample and CLIP containing molecular beacons detecting EGFR L858R and T790M were loaded onto a droplet chip for microfluidic-assisted fusion. Pump pressures were maintained at 2500 mBar and 1100 mBar for the oil and aqueous phases, respectively. Droplets were collected in microcentrifuge tubes for 20 min and stored at 4°C until further analysis.

[0068] Examples 1-13: Statistical Analysis Experiments were performed independently at least three times, and the number of independent replicates used for each experiment is indicated in each figure legend. Comparisons between two groups were performed using a two-tailed unpaired student's t-test, and comparisons between three or more groups were performed using one-way ANOVA. All statistical analyses were performed using Graphpad Prism 9.3.1.

[0069] Example 2: High-throughput charge-tuning of liposomes In this study, we utilized two types of lipids, cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), as building blocks for the preparation of charged liposomes (CLIPs) to achieve highly efficient charge control of the liposomes. By utilizing microfluidic hydrodynamic focusing (MHF) for liposome formulation (Jahn, A., et al., J Am Chem Soc 2004, 126(9), 2674-5), we successfully altered the lipid composition and efficiently and rapidly tuned the surface charge of CLIPs (Figures 1a, 1b, and 2). The microfluidic device facilitated CLIP formation by flowing a lipid solution (composed of DOTAP and DOPC) dissolved in ethanol at a concentration of 5 mg / mL through the central inlet channel at a rate of 5 μL / min, and an aqueous solution through two side inlet channels at a rate of 50 μL / min, resulting in successful CLIP formation. Figure 1c shows that the round morphology of the liposomes confirmed by transmission electron microscopy (TEM) (diameter: 92.5 ± 5.65 nm) is consistent with the size measured by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) (Figure 3).

[0070] As shown in Figure 1d, the surface charge of CLIP was adjusted between -18.63 ± 0.76 mV and +38.67 ± 2.51 mV by varying the composition of the building block lipids, DOTAP and DOPC. Ideally, the higher the DOTAP ratio, the more positive the CLIP surface charge, and vice versa. By adjusting the DOTAP ratio of the lipids from 0% (pure DOPC) to 100% (pure DOTAP), we confirmed that the surface charge shifted significantly from negative to positive when 25% DOTAP was included in the total lipid content. This change was consistent with the predicted results, as the surface charge increased proportionally with increasing DOTAP ratio (Figure 1d). Notably, after 48 hours of storage, the size of liposomes containing 0%, 25%, 50%, and 75% DOTAP remained stable over time. However, the liposomes containing 100% DOTAP showed a continuous increase in size up to 300 nm, specifically, 311.50±8.51 nm, an approximately three-fold increase in size (FIG. 1e, FIG. 4).

[0071] Example 3: Charge-induced fusion of liposomes After fine-tuning the surface charge of liposomes, we sought to confirm their effect on extracellular vesicles and CLIP fusion. First, we used a lab-on-a-disc system with an internal nanofilter (ExoDisc) to isolate EVs from H1975 cells, a rapid, label-free, and high-yield method (Figure 5a). Using a tabletop centrifugal microfluidic system, we were able to enrich EVs with a size of 20-200 nm within 30 minutes using cell culture supernatant (CCS) as the starting material (Figure 5b). Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) confirmed that the size distribution of EVs was homogeneous (Figure 5c, Figure 5d).

[0072] Next, to ensure precise control over the stoichiometry of EVs (EVs and CLIP) and prevent undesired aggregation, the entire fusion process was carried out in a droplet reactor using a precisely controllable μencapsulator, minimizing potential aggregation that can occur in bulk-scale reactions (Figure 6). The μencapsulator operates by flowing EVs and CLIP in separate phases through two aqueous inlets at a flow rate of 1.5 μL / min, while a continuous oily phase inlet delivers the biocompatible surfactant FC-40 at a flow rate of 35 μL / min, enabling the formation of aqueous droplets containing two EVs when they meet at the junction within the oily phase (Figure 7a). The amount of EVs and CLIP contained in a single droplet could be controlled by adjusting the initial input concentration of EVs using Poisson distribution calculations (Table 1).

[0073] [Table 1]

[0074] In Table 1, 10 4 ~10 8 The probability of a droplet containing a specific number of EVs at a range of particles / mL concentrations is displayed as a percentage.

[0075] With this microfluidic setup, the initial input concentration of EVs (10–10) was obtained within a single droplet of 27.40 ± 2.06 μm size. 5The ratio of EVs (in the EV / µL range) to CLIP was controlled (Figure 8). As shown in Figure 7f, TEM analysis confirmed the regular morphology of both semi-fused and fused vesicles. DLS analysis also showed an increase in the size of the vesicle population after fusion, which is consistent with the observed results (Figure 7g). However, as the ratio of DOTAP to CLIP for EV fusion was gradually increased, the size of the fused vesicles remained unchanged until it reached 75% DOTAP, at which point the vesicle size increased significantly (Figure 9a). Confirmation of vesicle fusion was further supported by observing changes in the surface charge of fused vesicles using a zeta sizer. The increased zeta potential correlated with a higher ratio of DOTAP, confirming the occurrence of fusion (Figure 9b).

[0076] To visualize EV-CLIP fusion events, we performed confocal laser scanning microscopy (CLSM) imaging, revealing EVs with a green fluorescent dye (3,3'-Dioctadecyloxacarbocyanine perchlorate (DiO) dye (Thermofisher Scientific, V22886)) and CLIPs with a red fluorescent dye (Rhodamine-DHPE (Lissamine rhodamine B 1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt)). CLSM imaging demonstrates colocalization of the dye within the fused endoplasmic reticulum, consistent with the TEM images and DLS experiments (Figure 7d and Figure 7e).

[0077] To assess fusion efficiency, we utilized a Förster Fluorescence Resonance Energy Transfer (FRET)-based lipid mixing assay (Stryer, L.; Haugland, R.P., Proc Natl Acad Sci USA 1967, 58(2), 719-26). CLIP dual-displays nitro-2,1,3-benzoxadiazole-4-yl (NBD) as the donor and lissamine rhodamine B (Rho) as the acceptor. When these molecules are brought into close proximity, energy transfer occurs from the excited NBD donor to the Rho acceptor (Francois-Martin, C.; Pincet, F., Scientific Reports 2017, 7(1), 43860) (Figure 7b, Figure 7c). We first investigated the effect of dual-displaying lipids on the zeta potential of CLIP, which was shown to have no significant effect on surface charge (Figure 11).

[0078] Using FRET-based analysis, we observed an increase in the fusion percentage corresponding to the DOTAP percentage (Figure 7c). However, despite the highest fusion efficiency, CLIP containing 100% DOTAP exhibited instability and formed aggregates over time (Figure 4). When comparing the droplet reactor fusion method with bulk scale, potential aggregates were observed (Figure 12). Consequently, 75% DOTAP was selected as the formulation for subsequent experiments. CLSM imaging further confirmed the maximum fusion percentage (74.96 ± 2.63%) by adjusting the CLIP:EV ratio to 10:1 (Figure 7c).

[0079] Example 4: Digital detection of tumor-derived EV miRNAs After successfully demonstrating efficient fusion between CLIP and EVs, we focused on directly examining the fluorescence response during the fusion process between molecular beacon (MB)-encapsulated CLIP and tumor-derived EV (tEV) miR-21 using confocal laser scanning microscopy (CLSM) to evaluate the effectiveness of the charge-fusion strategy for detecting EV miRNA in its natural environment. In this study, the MB utilized a hairpin structure containing a cyanine 3 fluorophore at the 5' end, a tetrachlorofluorescein quencher at the 3' end, and a single-stranded oligonucleotide probe featuring complementary bases at both ends to bring the fluorophore and quencher into close proximity, thereby inducing fluorescence quenching (Zhang, P., et al., Angew Chem Int Ed Engl 2001, 40(2), 402-405) (Table 2).

[0080] [Table 2]

[0081] The underlined parts in Table 2 are the fluorophore and quencher, respectively. For the sequences, see Hu, J., et al., Biomaterials, 2018, 183:20-29.

[0082] First, we detected tumor-derived extracellular vesicles (tEVs) using molecular probes (MBs) specifically designed to target microRNA-21 (or miR-21), which is known to be upregulated in various tumor types, such as breast, colon, lung, pancreatic, prostate, and gastric cancers (Krichevsky, A.M.; Gabriely, G., J Cell Mol Med 2009, 13(1), 39-53). While generating CLIPs using the microfluidic hydrodynamic focusing method described above, miR-21-specific MBs (miR-21MBs) were loaded into the aqueous phase at 1 μM. This insertion did not significantly affect liposome size or zeta potential (Figure 13b). As a proof-of-concept, we fused these EVs with CLIPs containing miR-21MB in a droplet reactor and initiated the detection of EVs derived from the H1975 lung cancer cell line in phosphate-buffered saline (PBS). The concentration of H1975 EV was varied from 10 to 10 while the concentration of CLIP was kept constant throughout the experiment. 5 When the concentration was changed to EVs / μL and analyzed through a series of image processing, the number of droplets for which fluorescent signals were detected increased proportionally (Figures 14a-14e).

[0083] To evaluate system performance, we sought to confirm its ability to detect EGFR L858R and T790M mutations (Zhao, B.X., et al., Mol Med Rep 2015, 11(4), 2767-74) in the H1975 lung cancer cell line. To achieve this, we introduced EGFR L858R and T790M mutation-specific MB sequences (Hu, J., et al., Biomaterials 2018, 183, 20-29) by integrating 1 μM of each beacon into the aqueous phase via a microfluidic hydrodynamic focusing method. Notably, the inclusion of these beacons did not significantly affect CLIP size or zeta potential (Figure 13).

[0084] Next, we successfully detected these mutations using droplet reactors generated by the μencapsulator under the same experimental conditions as previously described, with the only difference being that CLIP contained specific beacons targeting the EGFR L858R and T790M mutations. Fluorescent signals were observed only in the presence of target EVs (Figure 15). Prior to detecting EGFR mutations in cancer patient plasma samples, we first demonstrated the detection of EGFR L858R and T790M mutations by introducing various concentrations of H1975-derived EVs into healthy human plasma (Figures 15b-15d). The concentration range was from 10 to 10 5 The detection results for the two mutations were consistent with those obtained with PBS (Figures 15e and 15f), confirming the robustness of the CLIP and EV fusion system. Remarkably, this system was able to detect levels as low as a single EV per 100 nanoliters, which is approximately 100-fold more sensitive than conventional methods (Figure 15d).

[0085] Example 5: Clinical applicability of EV-CLIP (charged-liposome EV detection) for patient sample analysis Finally, we attempted to validate the EV-CLIP system by testing EGFR L858R and T790M mutations in plasma clinical samples collected from lung cancer patients (Figure 17a). Plasma samples from 10 healthy donors and 73 lung cancer patients were included (Table 3).

[0086] [Table 3]

[0087] Table 3 shows basic information, including gender, age, histological morphology, smoking status, mutation, and disease stage, of the 10 healthy donors and 73 patients used in the examples. There were 10 healthy donors, 17 cancer patients without mutations (6 stage I, 3 stage II, 3 stage III, and 5 stage IV), and 56 cancer patients with mutations (18 stage I, 6 stage II, 7 stage III, and 25 stage IV). Samples were provided by Institutional Review Board-approved donors from Inha University Hospital, Chonnam National University Hwasoon Hospital, and Busan National University Hospital.

[0088] Among the lung cancer patients, 17 lacked mutations and 56 had mutations. The detection process was performed without any sample processing. Interestingly, the signals obtained from the plasma of cancer patients with both the L858R and T790M mutations were significantly higher than the critical values ​​derived from the ROI curves (Figure 17d). These results suggest a strong correlation between the presence of these mutations and the elevated signal levels of the EV-CLIP system. Regardless of the stage of cancer, only cancer patient samples with positive mutations exhibited significantly elevated signals. This EV-CLIP method can detect mutations at high levels in a short time without any processing, even in early-stage cancers, making it easy to distinguish cancer patients from healthy donors and enabling early diagnosis of drug resistance in cancer patients. [Industrial Applicability]

[0089] The charged-liposome EV detection (EV-CLIP) method of the present invention provides accurate quantification of rare EV subpopulations, opening up new avenues for exploring fundamental questions in cancer biology and providing highly useful clinical applications. This fusion system has great potential for diverse applications, including direct application in diagnostics, drug delivery vehicle development, EV marker profiling, and numerous other possibilities.

[0090] While the present invention has been described in detail above, it is obvious to those skilled in the art that the specific details are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0091] Sequence Catalog Free Text Electronic file attached.

Claims

1. A liposome for detecting cancer cell-derived extracellular vesicles (EVs), comprising a cationic lipid and a neutral lipid, The liposome is characterized in that a cancer cell-specific molecular beacon is encapsulated within the liposome.

2. The cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleyloxy-3-dimethylamino-propane (DODMA, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium bromide (DODAB), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and dioctadecyldimethylammonium. ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dimyristyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2(s Spermine carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest- -5-ene-3β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-KDMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (Dlin-MC3-DMA), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)- 1-propanaminium bromide (GAP-DMRIE), (±)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propanaminium-1-aminium (DOBAQ), 1,2-dimyristooyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane ( DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-aminium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl 2,3-bis(tetradecyloxy)propan-1-amine (DMDMA ), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino-ethyl)-amino]-ethylamino)propionamide (lipidoid 98N12-5) and 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200).

3. 2. The liposome for detecting extracellular endoplasmic reticulum according to claim 1, wherein the neutral lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA), and phosphatidylcholine (PC).

4. 2. The liposome for detecting extracellular endoplasmic reticulum according to claim 1, wherein the cationic lipid is 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

5. 2. The liposome for detecting extracellular endoplasmic reticulum according to claim 1, wherein the molar ratio (%) of the cationic lipid in the liposome is 25 to 75%.

6. A composition for detecting cancer cell-derived extracellular endoplasmic reticulum, comprising the liposome according to claim 1.

7. A kit for detecting cancer cell-derived extracellular endoplasmic reticulum, comprising the liposome according to claim 1.

8. A method for detecting extracellular vesicles derived from cancer cells, comprising the steps of: fusing the liposome described in claim 1 with extracellular vesicles derived from a biological sample; and determining that the extracellular vesicles are extracellular vesicles derived from cancer cells when a fluorescence signal is generated.

9. The method for detecting cancer cell-derived extracellular endoplasmic reticulum according to claim 8, wherein the fusion is performed in a droplet reactor comprising two aqueous phase channels, one oil phase channel, one junction, and one outlet channel.

10. The method for detecting cancer cell-derived extracellular vesicles as described in claim 9, characterized in that liposomes and extracellular vesicles derived from a biological sample are respectively introduced into the two aqueous phase channels, and aqueous droplets are formed at the junction.

11. A composition for diagnosing cancer, comprising the liposome of claim 1.

12. 12. The cancer diagnostic composition according to claim 11, wherein the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung cancer, peritoneal cancer, colon cancer, biliary tract tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, thyroid cancer, oral cancer, osteosarcoma, gallbladder cancer, biliary tract cancer, kidney cancer, bladder cancer, renal cell carcinoma, melanoma, brain cancer, glioma, glioblastoma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, small intestine cancer, esophageal cancer, colon cancer, stomach cancer, eye cancer, urethral cancer, cervical cancer, prostate cancer, ovarian cancer, metastatic cancer, head and neck cancer, rectal cancer, non-Hodgkin's lymphoma, multiple myeloma, acute myeloid leukemia, lymphoma, acute lymphoblastic leukemia, and chronic myeloid leukemia.

13. A cancer diagnostic kit comprising the liposome of claim 1.

14. 14. The cancer diagnostic kit of claim 13, wherein the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung cancer, peritoneal cancer, colon cancer, biliary tract tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, thyroid cancer, oral cancer, osteosarcoma, gallbladder cancer, biliary tract cancer, kidney cancer, bladder cancer, renal cell carcinoma, melanoma, brain cancer, glioma, glioblastoma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, small intestine cancer, esophageal cancer, colon cancer, stomach cancer, eye cancer, urethral cancer, cervical cancer, prostate cancer, ovarian cancer, metastatic cancer, head and neck cancer, rectal cancer, non-Hodgkin's lymphoma, multiple myeloma, acute myeloid leukemia, lymphoma, acute lymphoblastic leukemia, and chronic myeloid leukemia.

15. A method for providing information for cancer diagnosis, comprising the steps of: fusing the liposome described in claim 1 with extracellular vesicles derived from a biological sample; and determining that the cancer is present when a fluorescence signal is generated.

16. The information providing method according to claim 15, wherein the method is performed in a droplet reactor including two aqueous phase channels, one oil phase channel, one junction, and one outlet channel.

17. 16. The method for providing information for cancer diagnosis according to claim 15, wherein the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung cancer, peritoneal cancer, colon cancer, biliary tract tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, thyroid cancer, oral cancer, osteosarcoma, gallbladder cancer, biliary tract cancer, kidney cancer, bladder cancer, renal cell carcinoma, melanoma, brain cancer, glioma, glioblastoma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, small intestine cancer, esophageal cancer, colon cancer, stomach cancer, eye cancer, urethral cancer, cervical cancer, prostate cancer, ovarian cancer, metastatic cancer, head and neck cancer, rectal cancer, non-Hodgkin's lymphoma, multiple myeloma, acute myeloid leukemia, lymphoma, acute lymphoblastic leukemia, and chronic myeloid leukemia.

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