Bubble-encapsulated exosomes and their uses

Abstract

Exobubbles comprising a perfluorocarbon encapsulated by an extracellular vesicle and / or an exosome are described. Methods of preparing the exobubbles, methods of treating cancer using the exobubbles, and methods of imaging using the exobubbles are also described.

Description

BUBBLE-ENCAPSULATED EXOSOMES AND THEIR USESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 617,125, filed on January 3, 2024, which is incorporated herein by reference.BACKGROUND

[0002] Exosomes are cell-derived small extracellular vesicles that are naturally secreted by all types of cells and widely distributed in various biofluids. They carry a variety of key bioactive molecules (e.g., nucleic acids, proteins, growth factors, cytokines] from their parent cells and convey them to neighboring or even distant cells through circulation. First discovered by Harding and Pan in 1983, they were initially viewed as cellular junk and as a means of eliminating cell contents. In 2007, it was discovered that exosomes contain mRNAs and miRNAs, and the story changed; they began to attract interest as mediators of intercellular communication. They are now considered one of the most significant diagnostic biomarkers associated with diseases. Isolation techniques are disclosed in Bu et al., Exosomes: Isolation, Analysis, and Applications in Cancer Detection and Therapy, ChemBioChem 20 (4): 451-61

[2019] , Once isolated, exosomes can be modified to overcome natural limitations, resulting in "designer exosomes”. See Jafari et al, BioDrugs 34: 567-86 (2020). However, exosomes provide weak scattering properties, making them difficult to use for optical visualization.SUMMARY OF THE INVENTION

[0003] The invention provides a method for optical scaling of extracellular vesicles via internal refractive index changes. EVs are challenging to visualize in general optical settings due to their innately weak scattering properties. The inventors have described a visualization technique that optically manipulates EVs, serving as a platform to unveil new insights into EV- based liquid biopsy system. This platform consists of a stable bi-phasic and core-shell structure, created by encapsulating a perfluorocarbon liquid into the core of an EV, termed exobubbles. The exobubbles can be used in imaging and cancer treatment.

[0004] In one aspect the invention provides a material composition of an exobubble comprising a perfluorocarbon (PFC) that is encapsulated in an extracellular vesicle, an exosome, or a combination of thereof. In the composition of the invention, the extracellular vesicle and / or exosome is isolated from a mammalian cell, including a T cell, a macrophage, a NK cell, a stem cell, a genetically-engineered cell, or a combination of thereof. The PFC can be C5F12(perfluoropentane), CeFu (perfluorohexane), CsFnBr (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof.

[0005] In another aspect the invention is a method of preparing the exobubble composition described above. The method includes a first step of isolating extracellular vesicles and / or exosomes by, for example, filtration, chromatography, precipitation, centrifugation, or a combination of thereof. In a second step, a perfluorocarbon is encapsulated into the exosome or extracellular vesicle by gentle addition of the PFC to a solution of the exosomes or EVs. The mixture is then emulsified under either bath-type or probe-type sonication for at least 1 minute to obtain the exobubbles. A ratio of 10 - 100 pL PFC to 1 mL of exosome solution (z.e. 1 x 109- 1 x 1011particles / mL) is preferred. Afterward, the mixture was emulsified under sonication (bath-type or probe-type sonicator, 1 min) to obtain the exobubbles.

[0006] In another aspect the invention includes method of using the exobubbles of the invention. The methods include treating cancer or suppressing cancer metastasis in a mammal, particularly a human patient, after administering an effective amount of the exobubbles of the invention to the mammal or patient. In this method the exobubble is made from a CAR T cell or a stem cell.

[0007] In yet another aspect the invention includes a method of visualizing EVs, extracellular vesicles including encapsulating a perfluorocarbon liquid into the core of an EV (an exobubble). In a first step of the extracellular vesicles are isolated by filtration, chromatography, precipitation, centrifugation, or a combination of thereof. In a second step, a perfluorocarbon is encapsulated into the extracellular vesicle by gentle addition of the PFC to a solution of the EVs. The mixture is then emulsified under either bath-type or probe-type sonication for at least 1 minute to obtain the exobubble. A ratio of 10 - 100 p L PFC to 1 mL of EV solution (i.e. 1 x 109- 1 x 1011particles / mL) is preferred. Afterward, the mixture was emulsified under sonication (bath-type or probe-type sonicator, 1 min) to obtain the exobubbles.BRIEF DESCRIPTION OF THE FIGURES

[0008] The present invention may be more readily understood by reference to the following figures, wherein:

[0009] Fig. 1 is a schematic showing preparation of the exobubbles.

[0010] Fig. 2 is a schematic showing use of exobubbles as an integrated theranostic platform for image-guided therapy.

[0011] Fig. 3 shows an image of exobubble after perfluorohexane (PFH) is encapsulated in exosome samples.

[0012] Fig. 4 provides a graph showing the size distribution of exobubbles.

[0013] Figs. 5a & 5b provide graphs showing (a) the hydrodynamic size and (b) the size distribution of exobubbles.

[0014] Figs. 6 shows microscopic images of the exobubbles of the invention at 4x and 40x magnification.

[0015] Figs. 7a & 7b shows microscopic images of the exobubbles in the (a) absence or (b) presence of ultrasound.

[0016] Figs. 8a & 8b show (a) in vitro ultrasound imaging of exobubbles and (b) quantification of ultrasound intensity of exobubbles. Error bars represent the standard deviation (n = 3).

[0017] Figs. 9a & 9b show ultrasound images observed by the Vevo3100 system before and after bubble-popping mode.

[0018] Figs. 10a - 1 Od show the characterizations of CAR-T cell-derived exobubbles made as described in Example 8.

[0019] Fig. 11 provides images showing in vitro time-lapse images of the CAR-T cell-derived exobubble treated murine pancreatic cancer cells.

[0020] Figs.12a & 12b show the cytotoxic effect of EVs and exobubbles after 24 h of cocultured with murine pancreatic cancer cells as described in Example 10. Panel (a): Culture with HEK 293 cell-derived EVs and exobubbles. Panel (b): Culture with CAR-T cell-derived EVs and exobubbles. Data are displayed as mean ± s.e.m. from biological quintuplicate.

[0021] Figs. 13a - 13g provide grams and images showing refractive index contrast mediates optical manipulation of EVs a, Schematics illustrate a visualization technique for the optical manipulation of EVs using low-refractive-index material. When the core refractive index is similar to the medium, incident light easily passes through the particles, resulting in weak scattering intensity. This makes observation challenging in a general optical setting. However, modifying the EV’s interior with a material possessing a lower refractive index than the medium induces strong scattering, allowing optical enlargement for improved visibility, b, Scattering efficiency spectra were calculated for EV by the diameter of core-shell particles and core refractive indices, c, Representative images of simulated scattered field intensity distributions for core-shell nanoparticles at different core refractive indices. Simulation results (b and c) are calculated with the following parameters: shell thickness; 4 nm; refractive index of shell; E39, and diameter of core-shell nanoparticles; 150 nm. d, Schematic of eVISONpreparation via PFH encapsulation, e, Scattering efficiency of bare EVs and eVISON. The scattering efficiency (relative to the PBS sample) was measured using a dark field microscope with a built-in spectrometer, f, Scattering efficiency was calculated for eVISON entirely filled with low-refractive-index material, g, Effective refractive index of eVISION. From these results (e-g), the PFH volume fraction in eVISION was estimated to be 0.42 and an effective refractive index of 1.299.

[0022] Figs.14a -14h provide graphs and images showing optical and physical characterization of eVISON. a, Representative refractive index tomogram of eVISON through maximum intensity projection (left) and measured refractive indices (right) of particles (N = 30). b, Reconstructed 3D refractive index tomograms (middle and right) of eVISION through 2D holograms, c Elemental mapping of the bare EV and eVISON by STEM-EDX. d, Optical microscopic images of bare EVs and eVISON. The differential interference contrast image shows the eVISON (bottom), but not the bare EV (top, right). The inserted schematic illustrates the conversion of an EV from an invisible state to a visible state via the droplet technique in an optical setting, e, Cryo-electron microscopy (Cryo-EM) images of the bare EVs (top, right) and eVISON (bottom). The inserted schematic shows that the physical size of the EV is maintained after optical scaling, f, Geometric (left), hydrodynamic (middle), and physical diameter (right) of the bare EVs an eVISON. The mean geometric (particle number, bare EV; N = 4, eVISION; N = 30), hydrodynamic (replicate number, bare EV; N = 3, eVISION; N = 3), optical diameter (particle number, eVISION; N = 90) were measured using cryo-EM, nanoparticle tracking analysis, and optical microscope, respectively, g, Geometric size distribution of the bare EVs and eVISON. The geometric size distribution of the particles was measured using an electrical sensing technique, h, The schematic represents eVISION's diameters.

[0023] Figs.15a - 15g provide graphs and images showing the ultrasensitivity of eVISON in fluorescence systems, a, In an eVISION (nc < nm), the strong scattering of light leads to sufficient emitted light through effective excitation of the fluorescence dye. b, Confocal microscopy images of Alexa Fluor (AF) 594-labeled EVs and eVISION. AF 594-NHS ester was used to stain the pan-proteins on the surface of EVs and was clearly visualized in EV droplets as a rim structure on the surface, c, Emitted field intensity distributions of bare EV and eVISION mimics with a dipole source (shell thickness; 4 nm, refractive index of shell; 1.39, dipole source; kern 630 nm). d, Light scattering distribution from bare EV and eVISION mimics using MiePlot. e, Imaging flow cytometry of bare EV and eVISION labeled with Phycoerythrin (PE) anti-human CD63 Antibody. The inserted images show bright field (BF),side scattering (SSC), and fluorescence (FL) imaging of the bare EV and eVISION, f, Scattering efficiency spectra were calculated for EV as a function of the core refractive index and wavelength of the incident light, g, Quantification results of flow cytometry from bare EV and e VISION labeled with various anti-human CD63 antibodies (pacific blue, AF488, PE, AF594, or AF647-conjugated antibodies).

[0024] Figs.16a - 16e provide graphs and images showing performance of eVISON. a, Schematics illustrating the droplet technique applicable to various EV labeling methods, including surface protein labeling, gene transfection, chemical insertion, and internal nucleic acid labeling, b, Quantification of flow cytometry results from bare EV and eVISION, c, Photographs of pan-protein and internal nucleic acid measurement from bare EV and eVISION by confocal microscope, d, Multiplexed protein biomarkers (CD63, PD-L1, and EGFR) measurement in bare EVs and eVISION in a microfluidic chip, e, Examination of singular or fused state of eVISION using a confocal microscope. After the droplet technique was applied, 80% of EVs were maintained in a singular state.DETAILED DESCRIPTION OF THE INVENTION

[0025] Exobubbles exhibit unique features: growth dynamic property, high internal pressure, echogenic signal, and a19F magnetic resonance (MR) signal. Exobubbles can be used for ultrasound imaging and19F MR imaging of various organs, including the heart, liver, pancreas, and brain. Because exobubbles collapse in response to ultrasound, the versatile theragnostic platform disclosed here could become a therapeutic option via cavitation-induced cell rupture. In sum, exobubbles have great potential in the image-guided therapy of cancer. Figure 2 provides a schematic showing the use of exobubbles as an integrated theranostic platform for image-guided therapy.Definitions

[0026] As used herein, a subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age. Thus, adult, juvenile, and newborn subjects are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject afflicted with a disease or disorder (e.g. Alzheimer’s disease). The term patient or subject includes human and veterinary subjects.

[0027] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or an adverse effect attributable to the disease. "Treatment," asused herein, covers any treatment of a disease in a mammal, particularly in a human, and can include inhibiting the disease or condition, i.e., arresting its development; and relieving the disease, i.e., causing regression of the disease.

[0028] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualify the amount of an agent which will achieve the goal of improvement in disease severity and the frequency of incidence over treatment of each agent by itself, while avoiding adverse side effects typically associated with alternative therapies. The effectiveness of treatment may be measured by evaluating a reduction in symptoms.

[0029] As used herein, "a delectably effective amount" of an exobubble is defined as an amount sufficient to yield an acceptable image using equipment which is available for clinical use. A detectably effective amount of the exobubbles may be administered in more than one injection. The detectably effective amount of the exobubbles can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, and the dosimetry. Detectably effective amounts of the exobubbles can also vary according to instrument and film-related factors. Optimization of such factors is well within the level of skill in the art. Ultimately, the attending physician will decide the amount of exobubbles to administer to each individual patient and the duration of the imaging study.

[0030] "Diagnosis" as used herein generally includes determination as to whether a subject is likely affected by a given disease, disorder or dysfunction. The skilled artisan often makes a diagnosis on the basis of various symptoms and / or one or more diagnostic indicators, i.e., a biomarker, the presence, absence, or amount of which is indicative of the presence or absence of the disease, disorder or dysfunction.

[0031] "Prognosis" as used herein generally refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis of a patient is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. It is understood that the term "prognosis" does not necessarily refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, the skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] As used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" also includes a plurality of such samples and reference to "the exobubble" includes reference to one or more exobubbles, and so forth.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.Exobubble Compositions

[0035] One aspect of the present invention provides an exobubble comprising a perfluorocarbon encapsulated by an extracellular vesicle and / or an exosome.

[0036] Extracellular vesicles (EVs) are lipid bound cell membrane-derived vesicles secreted by cells into the extracellular space. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are differentiated based upon their biogenesis, release pathways, size, content, and function. Exosomes (30-400 nm) are produced by the endosomal pathway, whereas microvesicles (100 nm-1 pm) and apoptotic bodies (1-4 pm) are produced by direct shedding from the plasma membrane. The content of EVs consists of lipids, nucleic acids, and proteins, and in particular proteins associated with the plasma membrane, cytosol, and those involved in lipid metabolism. Zhang et al., Cell Biosci., 9: 19 (2019). Likewise, exosomes are small, single-membrane, secreted organelles of ~30 to ~200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Pegtel D. and Gould, S., Annu Rev Biochem, 88:487-514 (2019).

[0037] Perfluorocarbons or PFCs, are organofluorine compounds that typically follow the formula CxFy, meaning they contain only carbon and fluorine; i.e., all C-H bonds have been replaced by C-F bonds. Perfluorocarbons include perfluoralkanes and perfluoroaromatic compounds. In some embodiments, the perfluoroalkenes are saturated. Perfluorocarbons arehighly stable because carbon- fluorine bonds are very strong, and have a high density; over twice that of water. Examples of perfluorcarbons include carbon tetrafluoride, perfluorooctane, perfluoro-3-methylpentane, perfluoro- 1,3-dimethylcyclohexane, and perfluorodecalin. Note that as used herein, perfluorocarbons also include organic compounds in which a small percentage (15% or less) of the fluorine atoms have been replaced with a different halogen atom such as chlorine or bromine.

[0038] In some embodiments, perfluorocarbons having from 4 to 12 carbons are used. In further embodiments, the perfluorocarbons have from 5 to 10 carbons. In some embodiments, the perfluorocarbon is selected from C5F12 (perfluoropentane), CeFu (perfluorohexane), CsFnBr (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof.

[0039] The perfluorocarbon is encapsulated by the exosome. In other words, the interior of the exobubble comprises perfluorocarbon, while the exterior of the exobubble comprises the exosome material. The presence of the perfluorocarbon within the exosome increases the size of the exobubble as compared with the original exosome. The exobubble typically has a diameter from about 50 nm to about 150 nm greater than the original exosome.

[0040] The exobubbles can have various diameters, depending on the size of the source vesicles. In some embodiments, the exobubble have an average diameter of about 100 nm to about 5 pm. In further embodiments, the exobubbles have an average diameter of about 150 nm to about 800 nm, while in yet further embodiments the exobubbles have a diameter from about 200 to about 600 nm.

[0041] The extracellular vesicles and / or exosomes used to prepare the exobubbles can be obtained from a variety of different types of cells. Types of cells from which vesicles can be obtained include mammalian cells, plant cells, bacterial cells, and yeast cells. In some embodiments, the extracellular vesicles and / or exosome is isolated from a mammalian cell selected from the group consisting of T cells, macrophages, NK cells, stem cells, and genetically-engineered cells, or a combination of thereof. Genetically-engineered cells are cells that have foreign DNA introduced which results in a stable genomic change to the cell. In some embodiments, the extracellular vesicle and / or exosome is isolated from a CAR-T cell. Chimeric Antigen Receptor (CAR) T cells are T cells that have been genetically modified to express a chimeric antigen receptor that is designed to recognize and bind to a specific antigen, typically on the surface of cancer cells.

[0042] Extracellular vesicles and / or exosomes can include a wide variety of lipids, proteins, and nucleic acids, based on the type of cell from which they are derived. However, in some embodiments, additional materials are added to the extracellular vesicles and / or exosomes. Examples of materials that can be added to extracellular vesicles and / or exosomes used to prepare exobubbles include imaging agents and therapeutic agents.

[0043] Examples of imaging agents include near infrared imaging agents, positron emission tomography imaging agents, single-photon emission tomography agents, fluorescent compounds, radioactive isotopes, and MRI contrast agents.

[0044] Therapeutic agents can also be included in the exobubbles. Examples of therapeutic agents include antimicrobial agents, anti-inflammatory agents, immune agents, and anticancer agents. In some embodiments, the exobubble further comprises an anticancer agent.Methods of Preparing Exobubbles

[0045] Another aspect of the invention provides a method of preparing an exobubble. The method includes the steps of: (1) isolating an extracellular vesicle and / or exosome from a mammalian cell; (2) encapsulating a perfluorocarbon into the extracellular vesicle and / or exosome. The mammalian cells can include any of the mammalian cells described herein. In some embodiments, the mammalian cell is a T cell, a macrophages, an NK cell, a stem cell, or genetically-engineered cell.

[0046] A variety of methods are known to those skilled in the art for isolating extracellular vesicles. In some embodiments, exosomes can be isolated by ultracentrifugation-based methods. Tauro et al., Methods, 56:293-304 (2012). Additional methods have been developed based on isolation by size, immunoaffinity capture, and precipitation of exosomes, including density gradient isolation, precipitation kits, Exosome Isolation kits (e.g., ExoMir® Kit, Biotium™) Immunoprecipitation, Multiplexed ExoSearch Chip, and Acoustic Nanofilter. Doyle L. and Wang M., Cells, 8(7): 727 (2019). In some embodiments, the extracellular vesicle and / or exosome is isolated from a cell using filtration, chromatography, precipitation, centrifugation, or a combination of thereof. For example, exosomes can be isolated from HEK293T cells using exoEasy Maxi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.

[0047] The inventors prepared exobubbles by physically encapsulating perfluorocarbon-based gas precursors into the hydrophobic part of the exosomes. The perfluorocarbon-based gas precursors, C5F12 (perfluoropentane) or CSFM (PFH, perfluorohexane) can be used, but the gas type can include other compounds with the formula of CsFnBr (perfluorooctyl bromide) andC10F20O5 (perfluoro-15-crown-5-ether). Accordingly, in some embodiments, the perfluorocarbon is selected from C5F12 (perfluoropentane), CeFi4 (perfluorohexane), CsFnBr (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof.

[0048] The perfluorocarbon can be encapsulated by the extracellular vesicles and / or exosomes to prepare the exobubbles using a sonication method. Briefly, the perfluorocarbon is added to a solution including the exosomes and / or extracellular vesicles. The exosome solution is then emulsified to for the exobubbles. For example, PFH (10 - 100 pL) was gently added to 1 mL of exosome solution (1 x 109- 1 x 1011particles / mL). Afterward, the mixture was emulsified under sonication (bath-type or probe-type sonicator, 1 min) to obtain the exobubbles.Methods of Treating Cancer

[0049] Another aspect of the invention provides a method of treating cancer in a subject. The method includes administering a therapeutically effective amount of exobubbles comprising a perfluorocarbon encapsulated by an extracellular vesicle and / or an exosome. The exobubbles used in the method of cancer treatment can have any of the characteristics of exobubbles described herein. For example, in some embodiments, the exobubbles include a perfluorocarbon that is selected from C5F12 (perfluoropentane), CeFu (perfluorohexane), CgFi ?Br (perfluorooctyl bromide), C10F20O5 (perfluoro- 15-crown-5-ether), or a combination of thereof. In some embodiments, the subject being treated has been diagnosed with cancer.

[0050] Cancer is a disease of abnormal and excessive cell proliferation. Cancer is generally initiated by an environmental insult or error in replication that allows a small fraction of cells to escape the normal controls on proliferation and increase their number. The damage or error generally affects the DNA encoding cell cycle checkpoint controls, or related aspects of cell growth control such as tumor suppressor genes. As this fraction of cells proliferates, additional genetic variants may be generated, and if they provide growth advantages, will be selected in an evolutionary fashion. Cells that have developed growth advantages but have not yet become fully cancerous are referred to as precancerous cells. Cancer results in an increased number of cancer cells in a subject. These cells may form an abnormal mass of cells called a tumor, the cells of which are referred to as tumor cells. The overall amount of tumor cells in the body of a subject is referred to as the tumor load. Tumors can be either benign or malignant. A benign tumor contains cells that are proliferating but remain at a specific site and are often encapsulated. The cells of a malignant tumor, on the other hand, can invade and destroy nearby tissue and spread to other parts of the body through a process referred to as metastasis.

[0051] Cancer is generally named based on its tissue of origin. There are several main types of cancer. Carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. In some embodiments, the cancer is selected from the group of cancer types consisting of sarcoma, carcinoma, and lymphoma.

[0052] Cancer can also be characterized based on the organ in which it is growing. Examples of cancer characterized in this fashion include bladder cancer, prostate cancer, liver cancer, breast cancer, colon cancer, and leukemia. Solid tumors are a solid mass of cancer cells that grow in organ systems, as understood by those skilled in the art, and are more associated with the formation of an immune suppressive tumor microenvironment. In some embodiments, the cancer being treated a solid tumor cancer selected from the group consisting of breast, colon, bladder, prostate, and lung cancer.

[0053] The effectiveness of cancer treatment may be measured by evaluating a reduction in tumor load or decrease in tumor growth in a subject in response to the administration of the modified immune suppressor cells. The reduction in tumor load may be represent a direct decrease in mass, or it may be measured in terms of tumor growth delay, which is calculated by subtracting the average time for control tumors to grow over to a certain volume from the time required for treated tumors to grow to the same volume.

[0054] Exobubbles can be used as therapeutic agents in cancer therapy. Outstanding therapeutic effects for various types of tumors can be expected due to the characteristics of Exobubbles that collapse by responding to ultrasound. Bursting exobubbles can cause cavitation-induced cell rupture, which will induce the dying cells' release of damage- associated molecular patterns (DAMPs). DAMPs are then recognized by immune cells and promote immunogenic cell death. For example, DAMPs can mature dendritic cells and activate cytotoxic T cells. As such, using exobubbles (for cell rupture) can enhance the anti-tumor efficacy of immune checkpoint inhibitors. In addition, after preparing specific EVs into exobubbles, they can directly deliver cytotoxic cargos at the targeted disease site. For example, EVs derived from CAR-T cells can target specific cells, as demonstrated in the Examples provided later herein.

[0055] Exobubbles can also be burst by applying a focused ultrasound beam, to deliver therapeutic agents. Accordingly, in some embodiments, ultrasound is applied to burst the exobubbles in the subject. The agent delivery is enhanced, because burst Exobubbles release shock waves (cavitation) to permeabilize cell membrane (sonoporation). In particular, targeted delivery of exobubbles derived from chimeric antigen receptor (CAR) T cells induces the immunogenic cell death of cancer cells via pyroptosis and necroptosis. Consequently, exobubbles can ameliorate the anti-tumor immunity of immune checkpoint inhibitors.

[0056] Exobubbles can have a direct anticancer effect based on DAMPs and the associated immune response. However, exobubbles can also exhibit targeting of tumor cells based on the content of the extracellular vesicle and / or exosome, or can be modified to include an anticancer agent. When the extracellular vesicle and / or exosome is isolated from a cell that has an affinity for cancer cells based on proteins or other materials present in the extracellular vesicle and / or exosome, the exobubbles prepared from those extracellular vesicles and / or exosomes will retain that affinity. An example of a cell having affinity for cancer cells is CAR-T cells. Accordingly, in some embodiments, the exobubble includes the extracellular vesicle and / or exosome that is isolated from a CAR-T cell.

[0057] In other embodiments, the exobubble further comprises an anticancer agent. A wide variety of anticancer agents are known to those skilled in the art. Preferably the anticancer agent is a hydrophobic anticancer agent that can easily associate with the exobubble. Examples of anticancer agents include angiogenesis inhibitors such as angiostatin KI -3, DL-a- difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (i)-thalidomide; DNA intercalating or cross-linking agents such as bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cisplatin, melphalan, mitoxantrone, and oxaliplatin; DNA synthesis inhibitors such as methotrexate, 3-Amino-l,2,4-benzotriazine 1,4- dioxide, aminopterin, cytosine -D-arabinofuranoside, 5-Fluoro-5’-deoxyuridine, 5- Fluorouracil, gaciclovir, hydroxyurea, and mitomycin C; DNA-RNA transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin; enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenz- imidazole 1- -D-ribofuranoside, etoposine, formestane, fostriecin, hispidin, cyclocreatine, mevinolin, trichostatin A, tyrophostin AG 34, and tyrophostin AG 879, Gene Regulating agents such as 5-aza-2’-deoxycitidine, 5-azacytidine, cholecalciferol, 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, all trans-retinal, all trans retinoic acid, 9-cis-retinoic acid, retinol, tamoxifen, and troglitazone; Microtubule Inhibitors such as colchicine, dolostatin 15,nocodazole, paclitaxel, podophyllotoxin, rhizoxin, vinblastine, vincristine, vindesine, and vinorelbine; and various other antitumor agents such as 17-(allylamino)-17- demethoxygeldanamycin, 4-Amino-l,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide, luteinizing-hormone-releasing hormone, pifithrin-D , rapamycin, thapsigargin, and bikunin, and derivatives thereof.

[0058] Methods of cancer treatment using the exobubbles described herein can further include the step of ablating the cancer. Ablating the cancer can be accomplished using a method selected from the group consisting of cryoablation, thermal ablation, radiotherapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high intensity focused ultrasound, photodynamic therapy, administration of monoclonal antibodies, and administration of immunotoxins.Methods of Imaging

[0059] Another aspect of the invention provides a method of imaging. The method includes delivering a detectably effective amount of exobubbles to a sample or tissue region, and visualizing the exobubbles in the sample or tissue region, wherein the exobubbles comprise a perfluorocarbon encapsulated by an extracellular vesicle and / or an exosome. The exoubbles of the present invention provide robust scattering properties that allow the ultrasensitive detection of biomolecules.

[0060] Exobubbles can be used as contrast agents for imaging methods such as ultrasound and 19F MRI to image various organs, including the heart, liver, pancreas, lung, and brain. Exobubbles make versatile contrast agents for molecular imaging (ultrasound and magnetic resonance (MR) imaging). The exobubbles with echogenicity and MR properties can be used in laboratories at universities, research institutes, and hospitals where basic and applied research are needed to detect various diseases. For example, one can generate exobubbles using extracellular vesicles from chimeric antigen receptor (CAR) T cells to target specific tumor cells. Alternately, exobubbles that use extracellular vesicles from stem cells can target inflammation. Exobubbles can also be prepared from cells that have been genetically modified to express a receptor or antibody having an affinity for other cells of interest in order to specifically target those cells.

[0061] The exobubbles used in the method of imaging can be any of the exobubbles described herein. For example, in some embodiments, the perfluorocarbon included in the exobubble is selected from C5F12 (perfluoropentane), CeFu (perfluorohexane), CsFnBr (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof. Likewise, insome embodiments, the extracellular vesicles and / or exosome of the exobubble is isolated from a mammalian cell selected from the group consisting of T cells, macrophages, NK cells, stem cells, and genetically-engineered cells, or a combination of thereof

[0062] The method includes delivering a detectably effective amount of exobubbles to a sample or tissue region, allowing the method to be used to image both ex vivo samples and in vivo tissue regions. Samples include, for example, cell cultures and biopsy samples. Tissue regions, on the other hand, refer to a portion or region of a subject, such as an arm, an abdomen, or a leg, or an organ within the subject such as the heart, liver, pancreas, lung, or the brain.

[0063] The exobubbles can be used in a variety of different types of imaging methods. Methods of imaging include ultrasound, magnetic resonance imaging, flow cytometry, optical imaging, positron imission tomogrpahy, and computed tomography imaging. In some embodiments, the exobubbles are visualized using fluorescent imaging. Examples of optical imaging include coherent optical imaging, bright-field microscopy, dark-field microscopy, phase-contrast microscopy, and holography.

[0064] Methods of imaging also include the step of visualizing the exobubbles in the sample or tissue region. Visualization is taking the data obtained using the imaging method and creating a visual image of the data that can be interpreted by the user. Visualization often includes computer processing of the data obtained using the imaging method.

[0065] In some embodiments, in addition to visualizing the exobubbles, the exobubbles are quantified. Exobubbles can be quantified using ultrasound imaging, wherein the image contrast is directly proportional to the exobubble concentration, or by optical microscopy, wherein individual exobubbles can be resolved due to their high optical signal, allowing for direct enumeration of the exobubbles.

[0066] EVs have been extensively studied as a vehicle for system-wide cellular interactions and for use as potential biomarkers for a variety of different diseases. Hu el al., Clin Cancer Res., 14(19):6246-52 (2008). Based on the proteins and other materials present in the lipid surface of the exobubble, the exobubbles may have an affinity for certain tissue or tissues, allowing them to aggregate in the tissue for which they have affinity. In some embodiments, the tissue for which they have affinity is pathological tissue, such as cancer.

[0067] Circulating extracellular vesicles (EVs), tiny membrane-bound particles (average diameter; 150 nm) released by cells, are gaining traction as a promising diagnostic tool in clinical settings. These EVs are plentiful and stable in biofluids, containing significant amounts of biomolecules (e.g., proteins, nucleic acids, lipids) unique to the parent cell in their surfacemembrane or in their core. Therefore, investigating EVs offers the potential for early disease diagnosis and valuable insights for monitoring disease progression and therapeutic response. Accordingly, in another aspect, the unique nature of the exobubbles of the invention allows for the imaging (e.g., ultrasound or MR) of various diseases, including cancer. The imaging can be used to guide therapy, or it can be used to obtain a diagnosis or prognosis regarding a disease, such as cancer.

[0068] Examples have been included to more clearly describe a particular embodiment of the invention and its associated cost and operational advantages. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular examples provided herein.EXAMPLESExample 1: Preparation of exobubbles

[0069] As illustrated in Figure 1, exosomes isolated from HEK293T cells were subjected to sonication by adding a perfluorocarbon to the exosome solution as described above. Then the mixture was emulsified under sonication using either a bath-type or a probe-type sonicator for 1 minute to obtain the exobubbles. Figure 3 shows an image of exobubble after perfluorohexane (PFH) is encapsulated in exosome samples.

[0070] Result: No substantial difference in transparency was observed in exosome solution (PBS, pH 7.4) before and after the addition of PFH in the absence of ultrasound. Exobubble solution, in which PFH is encapsulated in exosomes, showed the opaque suspension.Example 2: Exobubble size distribution measurement

[0071] The size distribution of the exobubbles made as described in Example 1 was measured using a nanoparticle tracking analysis system (NanoSight LM10; Malvern Instruments, Worcestershire, UK). See Figure 4.

[0072] Result: The size distribution indicated that PFH encapsulation into exosomes increased their hydrodynamic size (Exosome = 172.48 ± 99.36 nm, Exobubble = 263.73 + 160.63 nm).Example 3: Measurement of hydrodynamic size and distribution

[0073] As shown in Figure 5, the hydrodynamic size and size distribution of the exobubbles created in Example 1 were measured using a dynamic light scattering system (Zetasizer Nano ZS; Malvern Instruments, Worcestershire, UK).

[0074] Result: The hydrodynamic size of Exobubbles (226.01 ± 98.95 nm) was significantly larger than that of bare exosomes (317.27 ± 78.42 nm) due to the PFH encapsulation.Example 4; Microscopy

[0075] The images of the exobubbles created in Example 1 were observed using an inverted microscope (Eclipse TE2000S; Nikon, Tokyo, Japan). See Figure 6.

[0076] Result: Bare exosomes were not observed in microscopic imaging, but Exobubbles were clearly observed. In particular, the Exobubbles had a spherical shape in an aqueous condition.Example 5; Microscopy in absence or presence of ultrasound

[0077] As shown in Figure 7, microscopic images of the exobubble created in Example 1 were taken in the (a) absence or (b) presence of ultrasound to investigate the morphological changes by adjusting frequency and exposure time. Then, time-dependent images of the exobubbles were observed using an inverted microscope (Eclipse TE2000S; Nikon, Tokyo, Japan).

[0078] Result: In the absence of ultrasound, Exobubbles maintained their structure for at least 180 s. However, the morphological images of Exobubbles were rapidly changed when we irradiated ultrasound. We also found that Exobubbles responded more dramatically to 3.3 MHz of ultrasound than 1 MHz at the same ultrasound power (2 W / cm2).Example 6: Observation of images using Vevo 3100 system

[0079] As shown in Figure 8 the exobubbles made in Example 1 were observed using the Vevo 3100 system (VisualSonics, Toronto, Canada). Panel (a) shows In vitro ultrasound imaging of an exobubble. Panel (b) shows the quantification of ultrasound intensity of the exobubble. Error bars represent the standard deviation (n = 3).

[0080] Result: We investigated the echogenic properties of Exobubbles in an agar-gel phantom. As expected, water and exosomes did not exhibit an ultrasound signal. In addition, the simple mixture of water and PFH generated a limited ultrasound signal because there was no PFH encapsulation material. Notably, a strong ultrasound signal was detected in Exobubbles, suggesting the potential of exosomes as a stable and robust shell material of ultrasound contrast agents.Example 7: Bubble pop imaging

[0081] The ultrasound images were observed by the Vevo3100 system before and after bubblepopping mode. This is shown in Figure 9. Panel (a) shows In vitro ultrasound imaging of the exobubble after popping. Panel (b)shown the quantification of ultrasound intensity of the exobubble. Error bars represent the standard deviation (n = 3).

[0082] Result: Due to the simple mixture of water and PFH generating no detectable ultrasound signals, there was no substantial difference in ultrasound intensity before and afterbubble-popping mode. Interestingly, after bubble popping mode, we have confirmed that the range of fluctuations in the ultrasound intensity by exobubble increased.Example 8: CAR-T cell-derived exobubbles

[0083] Figure 10 shows the characterizations of CAR-T cell-derived exobubbles made as follows. EVs were isolated from the culture media of Mesothelin scFv-TM8-4-lBB-CD3z CAR-T cells via size exclusion chromatography (IZON, Medford, USA). Perfluorohexane (CeFu, PFH) was then incorporated into the core of EVs. Specifically, 10 pL of PFH was added to 1 mL of EV solution (1 x 109- 1 x IO10EVs / mL), and the mixture was sonicated with a probe-type sonicator (30 s, twice).

[0084] Result: Figure 10 panel (a) shows a Western blot of EVs from HEK293 and CAR-T cells. CAR-T EVs contain key markers (CD8a, Perforin, scFv) that are present in CAR-T cells. HEK293 -derived EVs (negative control) were devoid of these markers. Both EV samples were positive for canonical EV markers (Alix, CD63). Panel (b) is a cryo-EM image of CAR-T cell- derived exobubbles (EBs). We observed no significant changes in EV size after encapsulating PFH. Scale bar, 50 nm. Panel (c) shows the quantitative ELISA analysis of granzyme B in EBs. CAR-T cell-derived Exobubbles were positive for this marker. Panel (d) shows the results of expression of perforin as measured via flow cytometry. CAR-T cell-derived EBs were positive for this marker. Data are displayed as mean + s.d. from biological triplicate.Example 9: Treatment of cancer cells with CAR-T exobubbles

[0085] Alexa Fluor-555-conjugated CAR-T Exobubbles were added to the murine pancreatic cancer cell lines for one hour. One set of samples (top) was treated with ultrasound (US) to burst Exobubbles. The other set (bottom) was performed without US to confirm the EVs’ own effect. Cells were monitored over time by confocal laser scanning microscopy. By comparing the cells' appearance of the two experimental sets over time, we identified that the therapeutic strategy using Exobubble and ultrasound is effective. Figure 11 shows In vitro time-lapse images of the CAR-T cell-derived exobubble treated murine pancreatic cancer cells. Blue and yellow indicate cancer cell nuclei and CAR-T cell-derived Exobubbles, respectively. Scale bar, 50 pm.

[0086] Result: Upon ultrasound treatment, CAR-T Exobubbles -treated cells displayed rapid changes in their morphology (green dashed circles) and underwent necrosis. This can be attributed to the release of perforins and granzymes in the CAR-T cell-derived EV. In contrast, an immediate cytotoxic effect was not observed in cells not treated with ultrasound. In thiscase, the cancer cells' abnormal cellular morphologies (red arrows) were monitored after 12 hours post-incubations.Example 10: Cytotoxic effects of EVs and exobubbles

[0087] To determine the selective effects of exobubbles (EBs), we conducted experiments based on the presence or absence of ultrasound treatment. As an additional control group, EVs EBs from HEK 293 cells were used. First, murine pancreatic cancer cells were treated with calcein AM, a cell -permeant dye that can be used to determine cell viability. The murine pancreatic cancer cells were then incubated with EVs or EBs. After 24-hour incubation, cell viability was determined based on the fluorescence intensity of calcein AM in the supernatant (released by dead cells). Calcein AM-treated cancer cells release the probes from the cytosol when the cells die. This experiment was designed to determine the cytotoxicity of HEK 293 cells and CAR-T cell-derived EVs or EBs based on the amount of dye leaked into the conditioned medium. The results are shown in Figure 12. Panel (a) shows images of the culture with HEK 293 cell-derived EVs and Exohubhles. Panel (b) shows images of the culture with CAR-T cell-derived EVs and Exobubbles. Data are displayed as mean ± s.e.m. from biological quintuplicate.

[0088] Results: HEK 293 cells derived EVs and EBs, even when at 5000 times higher in number than cancer cells, did not cause significant cell death. These results indicate negligible cytotoxicity of EVs and EBs, as well as ultrasound treatment. CAR-T cell-derived EVs and EBs exhibited cytotoxic effects in a concentration-dependent manner. For example, at the EV (or EB)-to-cell ratio of 1000, the observed cell-death rate was 30%; this rate increased to 40% when the EV(or EB)-to-cell ratio was 5000. Importantly, EBs’ cytotoxic effect increased markedly when ultrasound was applied to burst EBs. This can be attributed to the release of cytotoxic molecules from EBs.Example 11: Optical enlargement of extracellular vesicles via internal refractive index changes

[0089] Circulating extracellular vesicles (EVs), tiny membrane-bound particles (average diameter 150 nm) released by cells, are gaining attraction as a promising diagnostic tool in clinical settings. These EVs are plentiful and stable in biofluids, containing significant amounts of biomolecules (e.g., proteins, nucleic acids, lipids) unique to the parent cell in their surface membrane or their core. Therefore, investigating EVs offers the potential for early disease diagnosis and valuable insights for monitoring disease progression and drug response. While various methods like nanoflow cytometry, fluorescence microscopy, imaging flow cytometry,and plasmonics have been employed, creating a direct and practical approach to visualize EVs remains challenging. Despite the contradiction between the poor light scattering of nanoscale materials and the need for visualization, this study focuses on developing a methodology to visualize EVs.

[0090] A notable insight into light scattering by nanoscale particles reveals that, particularly for spherical particles in liquids, the scattering intensity is governed by the particle diameter and the refractive index ratio between the particle (np) and the surrounding medium (nm). In the case of EVs, primarily constituted of water in their intraluminal region (nWater = 1.333), the refractive index contrast with the surrounding medium is minimal. This results in subdued light scattering within general optical settings. Consequently, conventional methods of EV analysis have tended to overlook a substantial portion of the invisible EVs, observing only limited subsections. Therefore, this study explores whether the EVs can be made visible through optical manipulations that alter the refractive index inside the EV.

[0091] Here, we report on a visualization technique that optically manipulates EVs, serving as a platform to unveil new insights into EV-based liquid biopsy systems. This platform, extracellular vesicle droplets with optical manipulation (eVISION), consists of a stable bi-phasic structure, also referred to as a core-shell structure, created by encapsulating a perfluorocarbon (PFC) liquid into the core of an EV. Most importantly, we show that the choice of the PFCs fundamentally and dramatically influences the optical appearance of nanoscale eVISION, making them appear larger than their physical size. A significant refractive index contrast with the surrounding medium and minimal impact on the inherent properties of the EVs characterize an optimal PFC. By introducing the concept of light scattering enhancers, we developed a CeFi4 (perfluorohexane, PFH) encapsulated eVISION for detecting cancer biomarkers at the single EV level. This eVISION achieves excellent sensitivity and enables the detection of multiple markers in flow cytometry. Overall, this eVISION further solidifies the fundamentals and applications of clinical EV testing as a diagnostic platform by visualizing EVs.Principle of .eVISION in .optical scaling.

[0092] According to the general Mie theory, the scattering intensity is mediated by the refractive index between the particle and surrounding medium when considering spherical particles of the same size (Fig. 13a). This assertion can be substantiated by calculating the effective dipole moment (P), expressed as:

[0094] with the corresponding equation for the cross-sections for scattering (Qsc):represent the electric field, the particle's radius, the dielectric function of the particle and medium, and dimensionless size (radius of particle multiplied by the wave vector in medium), respectively. Given these equations (1) and (2), we can conclude that for nanoparticles of the same size, the scattering efficiency is proportional to A(np- nm)2. We performed nanophotonic simulations: The refractive index contrast of a component was systemically increased, influencing the scattering cross-section, thereby increasing scattering efficiency (Fig. 13b). This manipulation enabled the optical observation of the particle. To test this, we first confirmed that an increase in refractive index contrast leads to a higher scattering efficiency with the same particle size in a given direction to the incident light (Fig. 13c). Importantly, the core-shell nanoparticle in an aqueous condition (nm= 1.333), when filled with a significantly lower refractive index component (refractive index of core; nc= 1.252), exhibited a significantly higher magnitude of scattered field intensity (SFI) compared to the EV mimetic particle (nc= 1.333). Notably, despite the EV’s shell having a thickness of approximately -4 nm and a refractive index of -1.39 (ref), the scattering intensity remained low due to its interior being filled with water.

[0097] To realize the optical scaling of EVs, we developed eVISION by encapsulating PFH (n; 1.252) into the EVs (Fig. 13d). After evaluating various PFCs based on factors such as boiling point, refractive index difference from the surrounding medium, and impact on physical size; we selected PFH as the optimal candidate for eVISION. The scattering efficiency measured from eVISION was 4.14-fold higher than that of bare EV (Fig. 13e). Moreover, when the interior of eVISION was entirely filled with PFH, the scattering efficiency increased to 24.02 times that of bare EV (Fig. 13f). We estimated the PFH volume fraction, revealing that 42% of the EV's interior is filled with PFH, resulting in eVISION having an effective refractive index of 1.299 (Fig. 13g).Characterization of eVISION.

[0098] We systematically conducted optical and physical characterization of eVISIONs, placing a particular emphasis on their refractive index for optical scaling. Thus, the examination of the refractive indices for each eVISION is pivotal (Fig. 14a). The average refractive index of the eVISIONs, measured at 1.29, demonstrated strong alignment withsimulation results (Fig. 13g). The introduction of PFH into EVs underscored the emergence of a bi-phasic and core-shell structure, distinctly visible in an aqueous condition (PBS, pH 7.4). The 3D holotomographic images clearly depicted the spherical shape of eVISION, revealing a distinct EV shell and core components (Fig. 14b). Further investigations into eVISION's atomic composition were conducted through scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy (STEM-EDX), exposing the presence of fluorine atoms enveloped by EV's organic elements (Fig. 14c).

[0099] We then proceeded to directly visualize eVISION using a confocal microscope with a differential interference contrast mode, and its striking visibility was a notable feature, in contrast to the invisibility of EVs in the same environment (Fig. 14d). The substitution of PFH into the EV core triggered a transformative shift from invisibility to visibility via the droplet technique in an optical setting. To validate the optical scaling of eVISION without physical size changes, we employed cryogenic electron microscopy (cryo-EM), affirming the preservation of geometric diameter in EVs post-PFH encapsulation into their core (Fig. 14e). Following this, we measured the mean geometric, hydrodynamic, and optical diameters of the bare EVs and eVISION (Fig. 14f). Quantitatively, there were no significant differences in the mean geometric diameter between bare EV (153.0 ± 27.0 nm) and eVISION ( 170.3 ± 42.2 nm). However, the eVISION exhibited approximately 1.4 times larger mean hydrodynamic diameter than bare EVs, attributed to the inverse relationship between particle size (based on Brownian motion) and viscosity in the Stokes-Einstein equation (viscosity; water = 0.89 cP, PFH = 0.64 cP). Consequently, eVISION had an optical diameter of 1.24 ± 0.52 pm (Fig. 14f) with similar geometric size distribution to bare EVs (Fig. 14g), implying an optical enlargement of approximately 7.3 times their actual size (Fig. 14h).Ultrasensitivity of eVISION in fluorescence systems.

[0100] We comprehensively evaluated the performance of eVISION in fluorescence imaging and flow cytometry, specifically addressing the challenge of ultrasensitive detection of EVs. In fluorescence systems with eVISION, highly sensitive detection of EVs involves a two-step process: (1) primary scattering of excitation light to activate the fluorophore and (2) secondary scattering of emitted light from the fluorophore (Fig. 15a). Consequently, the superior sensitivity of EV detection with eVISION is directly proportional to the square of both the primary and secondary scattering efficiencies. The superior analytical performance of eVISION is demonstrated in Fig. 15b. We labeled the pan-protein of the EVs' membranes with fluorophores and imaged them under consistent conditions. Remarkably, eVISION detected asignificantly higher number of EVs compared to conventional EV imaging, with particle counts of 2298 (e VISION) and 49 (bare EV) in the same field of view (177 pm x 177 pm). The mean fluorescence intensity of a single eVISION particle was 1.15 times higher than that of the bare EV; however, the total fluorescence intensity (area x mean fluorescence intensity) of the entire particle was 8.73 times higher. These results are consistent with the 7.3-fold scaling of the optical size of eVISION (Fig. 14f). We further conducted an in-depth analysis of the emitted light distribution surrounding eVISION by mimicking the presence of fluorophores on its surface (Fig. 15c). This was demonstrated using nanophotonic simulation, and SFI images were generated by averaging each polarization state of the dipole source (kem= 630 nm). Notably, a significant amount of light (as secondary scattering) was presented throughout the particle, particularly in the region adjacent to the dipole source of the eVISION mimics, which exhibited the strongest SFI. The SFI of the eVISION mimics exceeded that of the bare EV mimics.

[0101] We subsequently validated the light scattering distribution from eVISION mimics using 360° MiePlot (Fig. 15d). The observed broader light scattering distribution in eVISION mimics, compared to bare EV mimics, involves assessing scatter along the path of the laser with forward scatter (FSC) and measuring scatter around 90° relative to the laser with side scatter (SSC). These unique characteristics of eVISION enable the effective utilization of conventional flow cytometry in EV analysis. We demonstrated that eVISION exhibits superior detection sensitivity in both imaging and conventional flow cytometers (Fig. 15e and sX). After labeling the CD63 of EVs with PE-conjugated antibody, eVISION detected more CD63+EVs than the conventional EV flow cytometry, with counted numbers of EVs at 2.92 x 104objects / mL (bare EV) and 9.62 x 106objects / mL (eVISION). The eVISION system also enhances selectivity by distinguishing true signals (from EV droplets with PFH) from unwanted signals. In EV analysis utilizing antibody-based immunostaining, eVISION consequently reduces the potential for false-positive signals arising from antibody aggregation (Fig. sX). Importantly, the advantages of e VIS ION extend beyond a specific light region and can be realized across a wide range of wavelengths in the 400-800 nm region (Fig. 15f)- As a result, eVISION provides the flexibility to choose fluorescence dye from various libraries for the detection of EVs (Fig. 15g).Performance of eVISION.

[0102] In evaluating the performance of eVISION, we harnessed its ultrasensitive capabilities in fluorescence systems to assess its potential for multiplexing various biomolecules in EVs. Various methods for labeling EVs exist, encompassing surface protein labeling, genetransfection, chemical insertion into lipid membranes, and internal cargo labeling (e.g., DNA and RNA); we initially applied eVISION to these methodologies (Fig. 16a). The application of eVISION revealed visible variations (compared to bare EV) in the number of detected EVs, leading to significant fold changes in flow cytometry: CD63-GFP+EV (73-fold), MemGlow+EV (10-fold), Pyronin Y+EV (317-fold), and Hoechst+EV (8-fold) (Fig. 16b). Moreover, we extended the use of eVISION to visualize RNA and DNA inside EVs. After labeling EVs with pan-protein (Cy5-NHS ester) and pan-nucleic acids (SYBR gold), eVISION exhibited significant signals for both proteins and nucleic acids, providing a clear visualization of nucleic acids inside EVs. Bare EVs, on the other hand, showed minimal signals (Fig. 16c). We categorized EVs into two groups: SYBR gold positive versus negative. The number of SYBR gold+EVs (i.e., EVs containing nucleic acids) was 200 per 1000 (Cy5+EVs; containing proteins).

[0103] Fig. 16d shows the ultrasensitive triple multiplexing capability of eVISION in a chip. Following the fabrication of eVISION (G1 i 36EGFR vUIcell-derived), we introduced three types of fluorescently conjugated antibodies and placed the mixture onto a (3- aminopropyl)triethoxysilane (APTES)-coated microfluidic chip. With eVISION, all three biomarkers (CD63, PDL1, and EGFR) were significantly more detected, whereas rare signals were observed in the absence of the droplet technique, resulting in particle counts of 2364 (eVISION) and 30 (bare EV) in the same field of view (177 pm x 177 pm). In multiplexing assay, eVISION exhibited more prominent triple-positive signals eVISION; 22.6%, bare EV; 0.7%) and double-positive signals (EV 14.0%, bare EV; 3.4%, EVCD63+EGFR+eVISION; 10.7%, bare EV;eVISION; 6.7%, bare EV; 0%). After applying eVISION to assess its multiplexing performance, we examined whether EVs exist in a singular or fused state (Fig. 16c). By mixing Gli36 / Z'77'’rinEVs (labeled with a green dye) and astrocyte EVs (labeled with a red dye), we generated eVISION under various fabrication conditions. The lower the external force (i.e., ultrasonic wave) applied to fabricate eVISION, the higher the proportion of EVs in the singular state (Fig. sX). Under optimized conditions (sonication power: 55 W, amplitude: 40%, pulse on: 5s, pulse off: Is, total: 60s), 80% of EVs were maintained in a singular state.Discussion

[0104] EVs remain elusive entities in conventional optical environments. The application of EV-based liquid biopsies represents an emerging frontier in cancer diagnostics; however, EVs' inherently weak scattering properties present a challenge to both direct observation andaccurate detection. We present an optical engineering technique, eVISION, designed to augment the capabilities and provide more informed clinical diagnoses. The eVISION platform facilitates direct visualization of EVs in general optical settings and enables ultrasensitive detection of biomolecules at the single EV level using fluorescence systems. The eVISION offers distinctive features and performance enhancements: (1) robust scattering properties that provide optical visibility (optical diameter: 1.24 pm); (2) apparent optical enlargement (>7.3- fold) without altering the original physical size; (3) superior detection sensitivity in fluorescence systems (approximately 330-fold higher than that of bare EV), achieved through the principle of the square of the first and second scattering efficiencies; (4) visualization of the core-shell structure, allowing distinct imaging of the proteins of the EV shell and internal nucleic acids; and (5) robust classification accuracy of -98% for patients with glioblastoma. These unique attributes derive from a fundamental physical principle: scattering intensity modulated by refractive index contrast for equivalent- size nanoparticles.

[0105] The amalgamation of quantitative analysis of biological constituents (e.g., proteins, RNA, DNA) and high-resolution structural visualization (core-shell structure) at the single EV level can open an innovative frontier in EV research. Nevertheless, regular fluorescence microscopes and flow cytometers confront limitations due to the diffraction limit of light (-200 nm). Beyond this inherent nature, e VIS ION holds the potential to explore fundamental questions regarding the origin of EVs by providing better information on EV-mediated intercellular communication as well as real-time snapshots of the molecular makeup of the tumor microenvironment. The PFCs used for optical manipulation have hydrophobic properties. The internal medium of EVs is partially replaced from water to hydrophobic PFCs, which inevitably leads to the loss of hydrophilic internal cargos such as nucleic acids. However, eVISION remarkably enhances biomarker-specific EV sorting efficiency (approximately 73- fold higher than bare EVs), complementing downstream analyses such as droplet digital polymerase chain reaction (ddPCR)-based genomic profiling.

[0106] The clinical implications of this optically-manipulated droplet technology are potentially wide-ranging. Further validation and development could position eVISION for clinical diagnostics, investigating EVs as cancer biomarkers, monitoring tumor response to therapy, and expanding its adaptability to diverse body fluids and diseases. Moreover, the simplicity of the eVISION manufacturing process (by sonication for 1 min prior to analysis) and the bioinert nature and commercial availability of the reagents used further enhance the potential of eVISION for clinical application. While the current study focused on glioblastomadiagnostics, the versatility of eVISION also extends to the development and validation of therapeutic EVs for broader research and clinical applications. Overall, based on the general Mie theory, optical size enlargement of nanoparticles through internal refractive index changes can provide fundamental insights into the study of various core-shell and mesoporous nanoparticles such as liposomes, lipid nanoparticles, silica nanoparticles, and polymeric nanoparticles.MethodsCell lines and cell culture

[0107] A human glioma cell line expressing epidermal growth factor receptor variant III (Gli36R'7A'l / / / ; RRID: CVCL_RL88) and a human embryonic kidney cell line expressing green fluorescent protein (HEK293T67A+) were cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen, Waltham, MA, USA) with high glucose, supplemented with 10% fetal bovine serum (FBS; Life Technologies Corporation, Carlsbad, CA, USA) and 1% penicillin / streptomycin solution (Pen / Strep; Life Technologies Corporation, Carlsbad, CA, USA). Immortalized human astrocytes (Astrocyte fetal-SV40; Applied Biological Materials Inc., Richmond, BC, Canada) were cultured in Prigrow IV medium containing 10% FBS, 2 mM L-glutamine, 10 ng / mL recombinant human EGF, and 1% penicillin / streptomycin. All experiments were conducted at 37°C in a humidified 5% CO2 atmosphere, maintaining cells at approximately 80% confluence.EV isolation from cell lines

[0108] We isolated EVs using the exoEasy kit (exoEasy Maxi Kit; Qiagen, Hilden, Germany) following the manufacturer's instructions and our previously established laboratory protocol. Initially, we seeded cells at a density of 2 x 106in 10 mL of growth media in a T75 flask and incubated cells for 24 h. Subsequently, after washing the cells with PBS, we replaced the media with EV-free media (3% exosome-depleted FBS and 1% Pen / Strep in DMEM or Prigrow IV medium). Following an additional 24 h incubation, we collected the media, which underwent centrifuging at 300 x g for 5 min at 25 °C. We collected the supernatant and applied a second centrifugation at 1200 x g for 20 min at 25°C, followed by filtration through a 0.8 pm filter to remove debris. Then, we isolated EVs from 10 mL of the conditioned media using the kit through the following steps: (1) mix 10 mL of conditioned media with binding buffer (1 / 1, v / v); (2) wash with an affinity column; (3) elute EVs in 1 mL aliquot; and (4) store the isolated EVs at -80°C.Preparation of eVISION

[0109] To prepare eVISION, we applied sonication to encapsulate PFCs within EVs. Five different PFCs were used in this study: perfluoropentane (PFP), PFH, perfluorooctane (PFO), perfluorodecalin (PFD), and perfluorophenantrene (PFPT). Initially, we placed 1 mL of EV (2 x 109to 2 x IO10particles / mL) in a low protein binding tube (ThermoFisher Scientific, Waltham, MA, USA). After adjusting the temperature of the EV solution and PFC to 4°C, we meticulously added 10 pL of PFC to ensure proper positioning at the bottom of the EV solution. Subsequently, we applied ultrasonic waves using a probe-type sonicator (QSONICA Q55; Qsonica Sonicators, Newtown, CT, USA; QSONICA Q125; Qsonica Sonicators, Newtown, CT, USA; or VCX500; Sonics & Materials, Inc., Newtown, CT, USA) for 1 min, which resulted in the turbid suspension of eVISION. We maintained the temperature of the resulting solution at 4°C before use.Optical imaging

[0110] To verify the optical diameter of the eVISION, we utilized a Nikon AX confocal microscope with a lOOx objective. We prepared different types of eVISION by individually encapsulating PFP, PFH, PFO, PFD, or PFPT. The volume of PFCs (10 pL per 1 mL of EV) was consistent for all samples. Samples were diluted 1:10 in PBS, and 10 pL of each sample was placed separately on the 35 mm glass bottom dish, then covered with a glass coverslip. We obtained the images in differential interference contrast mode without fluorescent labeling of eVISION. Finally, 90 particles were randomly selected from three images and analyzed using the built-in software.Microfluidic resistive pulse sensing

[0111] To assess the geometric diameter of eVISION, we used electrical sensing technology with the nCSl instrument (Spectradyne, Signal Hill, CA, USA). Samples, diluted 1:10 in PBS, were measured according to the manufacturer's instructions. A microfluidic cartridge (C-900) measuring 130 nm to 900 nm was used for all measurements.Nanoparticle tracking analysis (NTA)

[0112] To investigate the hydrodynamic diameter of eVISION, we performed NTA using NanoSight (LM10; Malvern Panalytical, Malvern, UK). The instrument had a 20x objective, a scientific CMOS camera, and a blue laser (X = 405 nm). Sample concentrations were adjusted from 1 x 10sparticles / mL to 1 x 109particles / mL. After introducing the samples into the module chamber, images were acquired with the following parameters: acquisition time of 60 s, shutter speed of 30 ms, screen gain set to 10, and detection threshold set to 5. The obtained images were then analyzed using the built-in software (NTA v3.1).Cryo-EM

[0113] We conducted cryo-EM imaging to examine the geometric diameter of eVISION with intact morphology. Samples were immobilized in vitreous ice using a cryofixing device (Vitrobot Mark IV System; ThermoFisher Scientific, Waltham, MA, USA). Briefly, 3 pL of the sample was placed on a copper mesh carbon grid (C-flat, 1.2 pm diameter holes, 1.3 pm spacing) and blotted for 4 s in a chamber maintained at 4°C with 100% humidity. Images were acquired with a Titan Krios G3i microscope (ThermoFisher Scientific, Waltham, MA, USA) operated at 300 kV.Holo tomographic imaging

[0114] The inventors conducted holotomographic imaging using a high-performance holotomography microscope (HT-X1; Tomocube, Daejeon, South Korea) to explore the refractive index of eVISION at the single-particle level. First, we applied 10 pL of eVISION to a 35 mm glass bottom dish and covered it with a glass coverslip. Utilizing the HT-X1, they obtained holotomographic images of eVISION, subsequently reconstructing them into 3D refractive index tomograms using the built-in software. The refractive index was calculated through the software's built-in functionality (i.e., regularization without NN condition), and further analysis was carried out using Image J (i.e., analyze particles function).Singular state analysis

[0115] To determine the singular or fused state of eVISION, we examined the co-localization ratio by mixing the two types of EVs and varying the ultrasonic conditions. We stained Gli36n; / 7'’vincell-derived EVs and astrocytes separately with different fluorescent dyes. G1 i 3 fEGFR vincell-derived EVs and astrocytes (2 x 1010particles / mL) were mixed with 100 pg of Alexa Fluor 488 and Cy5, respectively. After reaction for 12 h at 4°C, we used a centrifugal filter (Amicon®, 3 kDa MWCO; Sigma-Aldrich, St. Louis, MO, USA) to remove unbound dyes. We then prepared eVISION using the “pre-mixing strategy” or the “after-mixing strategy.” For the pre-mixing strategy, we mixed 0.5 mL of Alexa Fluor 488-labeled GH36EGFR vIII cell-derived EVs and 0.5 mL of Cy5-labeled astrocyte-derived EVs and then prepared eVISION to verify the single or fused state. For the after-mixing strategy (used as a positive control for singular state), two types of eVISION were prepared separately from Alexa Fluor 488-labeled GH36EGFR vIII cell-derived EVs or Cy5-labeled astrocyte-derived EVs and then mixed before imaging (see Supplementary Information). To generate eVISION, we added 10 pL of PFH to 1 mL of EV solution and applied ultrasonic waves for 1 min (pulse on; 5 s, pulse off; 1 s). Additionally, we utilized a 20 kHz ultrasound frequency and adjusted theultrasound power from 55 W to 500 W with amplitude from 20% to 40%. We diluted eVISION in PBS (1 in 10) to prevent self-aggregation at high concentrations. The eVISION was captured on an APTES-coated microfluidic chip. After a 45 min incubation at 4°C, the chip was washed thrice with PBS. Finally, fluorescence images were captured using a Nikon AX confocal microscope with a l OOx objective. Using Image I software (NIH), we processed the images through the background subtraction function and analyzed the co-localization ratio with Comdet v0.5.5.Fluorescence imaging

[0116] To investigate the ultrasensitivity of the eVISION, we utilized a Nikon AX confocal microscope with a lOOx objective. We stained Gli36GG7A’vIUcell-derived EVs with fluorescent dye. Gli36£G™vIUcell-derived EVs (2 x 1010particles / mL) were mixed with 100 pg of Alexa Fluor 594. After reaction for 12 h at 4°C, we used a centrifugal filter (Amicon®, 3 kDa MWCO; Sigma-Aldrich, St. Louis, MO, USA) to remove unbound dyes. Then, we prepared eVISION by encapsulating PFH (10 pL per 1 mL of EV). Sample was diluted 1: 10 in PBS, and 100 pL of eVISION was loaded onto an APTES-coated microfluidic chip. After a 45 min incubation at 4°C, the chip was washed thrice with PBS. Finally, fluorescence images were captured using a confocal microscope. Using Image J software (NIH), we processed the images through the "threshold function and analyzed them by the analyze particle function.Imaging flow cytometry

[0117] We analyzed EV subpopulations using eVISION on an imaging flow cytometer. First, we labeled the Gli36AGAA’vUIcell-derived EVs with a fluorescence-conjugated anti-human antibody. After blocking the EVs with an Fc receptor blocking solution (Human TruStain FcX; BioLegend, San Diego, CA) for 10 min at 25°C, the EVs were stained in the dark for 45 min at 25 °C. The following antibodies were used in this study: Pacific Blue™ anti-human CD63 antibody, Alexa Fluor® 488 anti-human CD63 antibody, PE anti-human CD63 antibody, Alexa Fluor® 594 anti-human CD63 antibody, Alexa Fluor® 647 anti-human CD63 antibody, and PE- conjugated anti-human IgG Fc antibody (see Supplementary Information). For internal nucleic acid labeling, we used Pyronin Y (ThermoFisher Scientific, Waltham, MA, USA) or Hoechst 33342 (ThermoFisher Scientific, Waltham, MA, USA), and the EVs were stained for 45 min at 25°C in the dark. After labeling, we purified excess antibodies and chemicals using a centrifugal filter (Amicon®, 100 kDa MWCO; Sigma-Aldrich, St. Louis, MO, USA). The labeled EVs were then resuspended in the original volume of PBS. We then prepared eVISION following the identical procedure described earlier (10 pL per 1 mL of EV). Results werequantified using the built-in software (IDEAS®) according to previously described methods. All gating strategies were consistently applied to the bare EV and eVISION samples.Multiplexed imaging

[0118] To investigate the ultrasensitive triple multiplexing capability of the eVISION, we utilized a microfluidic chip and Nikon AX confocal microscope. First, we prepared eVISION by encapsulating PFH (10 pL per 1 mL of EV) into Gli36£G™vincell-derived EVs. After blocking the eVISION with an Fc receptor blocking solution (Human TruStain FcX; BioLegend, San Diego, CA), we added Pacific Blue™ anti-human CD63 Antibody, PE antihuman CD63 antibody, and Alexa Fluor® 647 anti-human EGFR Antibody. Subsequently, we loaded 100 pL of mixture solution onto an APTES-coated microfluidic chip. After a 45 min incubation at 4°C, the chip underwent thrice washing with PBS to remove excess antibodies. Multiplexed fluorescence imaging was acquired using a confocal microscope with a lOOx objective. We processed the images through the background subtraction function and analyzed them using Comdet v0.5.5 in Image J software (NIH).Blood plasma processing

[0119] The pilot clinical study was approved by the Institutional Review Board (IRB) ethics committee at Massachusetts General Hospital. Additionally, we provided comprehensive information to all participants and obtained written informed consent from each of them. The initial step involved the collection of whole blood samples from patients, utilizing K2 EDTA tubes containing an inert gel barrier (BD Vacutainer, Franklin Lakes, NJ, USA). Subsequent to the blood collection, we performed centrifugation at 1100 x g for 10 min at 20°C to separate the plasma. The plasma sample was then carefully passed through a 0.8 pm filter, aliquoted to a volume of 1 mL, and stored at -80°C.EV sorting

[0120] To classify specific EVs from eVISION, we performed nanoscale sorting with the CytoFLEX SRT Cell Sorter (Beckman Coulter, Brea, CA, USA). The entire setup was executed following established methods (ref). We prepared bare EV and eVISION labeled with PE antihuman CD63 antibody. After dilution in PBS by the same factor, we conducted sorting of CD63+ EVs from the samples for 1 hour. The event numbers were recorded using the 561- 579 / 16 channel, and the sorted EVs were stored at -80 °C for further downstream analysis. ddPCR

[0121] For downstream analysis of eVISION, we extracted EV RNA and employed ddPCR. Frist, we used the Qiagen exoRNeasy kit (Qiagen, Hilden, Germany) to extract EV RNA,following the manufacturer’s instructions and our established laboratory protocol (Ref.). Then, we used the Agilent 6000 RNA Pico assay (Agilent Technologies, Santa Clara, CA) to quantify RNA size and concentration. For ddPCR, we converted the extracted RNA (14 pL) into complementary DNA (cDNA) using the SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA) with thermocycling, in accordance with the manufacturer’s instructions. In the ddPCR amplification, we added 2 pL of cDNA, 1 pL of GAPDH-VIC probe (Hs99999905_ml; ThermoFisher Scientific, Waltham, MA, USA), 1 pL of EGFR i7 / / -mutant probe (5'-FAM-TGACAGATCACGGCTC-MGBNFQ-3') (ref), 10 pL of ddPCR™ Supermix solution (no dUTP; Bio-Rad, Hercules, CA, USA), and 6.5 pL of distilled water (UltraPure; Invitrogen, Waltham, MA, USA). Subsequently, we generated droplets using the QX200 manual droplet generator (Bio-Rad, Hercules, CA, USA) with Droplet Generation Oil (BioRad, Hercules, CA, USA). Following thermocycling (ref), we incubated the plate for 15 min at 4 °C and quantified the counts using QuantaSoft (version 1.7.4; Bio- Rad, Hercules, CA, USA).Optical simulations

[0122] Finite-difference time-domain (FDTD) simulations (FDTD Solutions, Lumerical) were conducted on eVISIONs to explore their scattering properties. The scattering cross-section of the single core-shell nanoparticle was calculated by first obtaining its electric and magnetic scattered fields using a total-field scatterd-field method with respect to a / ^-polarized plane wave. Then, the scattering efficiency was defined as the ratio of its scattering cross-section to its physical cross-section (i.e., particle size). The background and shell indices of the simulation were set to 1.333 and 1.39, respectively. The core indices were set to xx, xx, and xx for PFH, xx, and xx, respectively. The thickness of the shell and the diameter of the core were set to 4 nm and 142 nm, respectively. Non-uniform grids with a spatial resolution of 1 nm for the shell and adjacent regions and 5 nm for the others were generated in the x-, y-, and ’-directions. Based on the simulation, the scattering field intensity and far-field distributions were obtained, as shown in Figs. 1c and 3d. Note that the results in Fig. 3c were obtained using a dipole source with a wavelength of 630 nm, placed 3 nm away from the shell in the -direction. The scattered intensity distribution was averaged for two dipoles oscillating in the xy- and xz-di rect ions. All other optical conditions were kept consistent with those for the plane wave.Dark field spectroscopy

[0123] The optical scattering properties of EVs were investigated using optical dark-field microscopy (Eclipse Ni-U, Nikon) equipped with a supercontinuum laser (Super K Compact,NKT Photonics) and a spectrometer (Maya2000 Pro, Ocean Optics). Aqueous droplets containing EVs were dropped onto a glass slide and covered with another slide glass, scattering intensity was measured in the backward direction, where light from the upper dark-field condenser illuminated the droplet and the scattered light was collected. The backward scattering efficiency was defined as the ratio of the spectra with EVs to those without EVs. The numerical aperture of the dark-field condenser objective lens for light incidence was xx, providing an angle of light incidence on the droplet ranging from xx to xx with respect to the surface normal.

[0124] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. An exobubble comprising a perfluorocarbon encapsulated by an extracellular vesicle and / or an exosome.

2. The exobubble of claim 1, wherein the perfluorocarbon is selected from C5F12 (perfluoropentane), CeFir (perfluorohexane), CsF Br (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof.

3. The exobubble of claim 1, wherein the exobubbles have an average diameter of about 200 nm to about 600 nm.

4. The exobubble of claim 1, wherein the extracellular vesicles and / or exosome is isolated from a mammalian cell selected from the group consisting of T cells, macrophages, NK cells, stem cells, and genetically-engineered cells, or a combination of thereof.

5. The exobubble of claim 1, wherein the extracellular vesicle and / or exosome is isolated from a CAR-T cell.

6. The exobubble of claim 5, wherein the exobubble further comprises an anticancer agent.

7. A method of preparing an exobubble, comprising:(1) isolating an extracellular vesicle and / or exosome from a mammalian cell;(2) encapsulating a perfluorocarbon into the extracellular vesicle and / or exosome.

8. The method of claim 7, wherein the extracellular vesicle and / or exosome is isolated from a cell using filtration, chromatography, precipitation, centrifugation, or a combination of thereof.

9. The method of claim 7, wherein the perfluorocarbon is selected from C5F12 (perfluoropentane), CoFi-i (perfluorohexane), CsFiyBr (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof.

10. The method of claim 7, wherein the mammalian cell is a T cell, a macrophages, an NK cell, a stem cell, or genetically-engineered cell.

11. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of exobubbles comprising a perfluorocarbon encapsulated by an extracellular vesicle and / or an exosome.

12. The method of claim 11, wherein the extracellular vesicle is isolated from a CAR- T cell.

13. The method of claim 12, wherein the exobubble further comprises an anticancer agent.

14. The method of claim 13, wherein ultrasound is applied to burst the exobubbles in the subject.

15. The method of claim 11, wherein the perfluorocarbon is selected from C5F12 (perfluoropentane), CeFu (perfluorohexane), CsFiyBr (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof.

16. A method of imaging, comprising delivering a detectably effective amount of exobubbles to a sample or tissue region, and visualizing the exobubbles in the sample or tissue region, wherein the exobubbles comprise a perfluorocarbon encapsulated by an extracellular vesicle and / or an exosome.

17. The method of imaging of claim 16, wherein the exobubbles are delivered to a tissue region.

18. The method of claim 16, wherein the exobubbles are visualized using coherent optical microscopy.

19. The method of imaging of claim 16, wherein the exobubbles are visualized using fluorescent imaging.

20. The method of imaging of claim 16, wherein the exobubbles are also quantified.

Images