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WO2025147567A1 - Exosomes encapsulés dans des bulles et leurs utilisations - Google Patents

Exosomes encapsulés dans des bulles et leurs utilisations Download PDF

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Publication number
WO2025147567A1
WO2025147567A1 PCT/US2025/010175 US2025010175W WO2025147567A1 WO 2025147567 A1 WO2025147567 A1 WO 2025147567A1 US 2025010175 W US2025010175 W US 2025010175W WO 2025147567 A1 WO2025147567 A1 WO 2025147567A1
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Prior art keywords
exobubbles
cell
evs
evision
cells
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Hakho Lee
Dong Gil You
Jueun Jeon
Leonora Balaj
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General Hospital Corp
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General Hospital Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
    • A61B6/02—Arrangements for diagnosis sequentially in different planes; Stereoscopic radiation diagnosis
    • A61B6/03—Computed tomography [CT]
    • A61B6/032—Transmission computed tomography [CT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
    • A61B6/02—Arrangements for diagnosis sequentially in different planes; Stereoscopic radiation diagnosis
    • A61B6/03—Computed tomography [CT]
    • A61B6/037—Emission tomography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
    • A61B6/48—Diagnostic techniques
    • A61B6/481—Diagnostic techniques involving the use of contrast agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/48—Diagnostic techniques
    • A61B8/481—Diagnostic techniques involving the use of contrast agents, e.g. microbubbles introduced into the bloodstream

Definitions

  • 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.
  • bioactive molecules e.g., nucleic acids, proteins, growth factors, cytokines
  • exosomes 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.
  • 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.
  • PFC perfluorocarbon
  • 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.
  • 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.
  • 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 10 9 - 1 x 10 11 particles/mL) is preferred. Afterward, the mixture was emulsified under sonication (bath-type or probe-type sonicator, 1 min) to obtain the exobubbles.
  • 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.
  • the exobubble is made from a CAR T cell or a stem cell.
  • the invention includes a method of visualizing EVs, extracellular vesicles including encapsulating a perfluorocarbon liquid into the core of an EV (an exobubble).
  • a perfluorocarbon liquid into the core of an EV (an exobubble).
  • 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 10 9 - 1 x 10 11 particles/mL is preferred.
  • the mixture was emulsified under sonication (bath-type or probe-type sonicator, 1 min) to obtain the exobubbles.
  • Fig. 1 is a schematic showing preparation of the exobubbles.
  • Fig. 2 is a schematic showing use of exobubbles as an integrated theranostic platform for image-guided therapy.
  • Fig. 3 shows an image of exobubble after perfluorohexane (PFH) is encapsulated in exosome samples.
  • PHF perfluorohexane
  • Fig. 4 provides a graph showing the size distribution of exobubbles.
  • Figs. 5a & 5b provide graphs showing (a) the hydrodynamic size and (b) the size distribution of exobubbles.
  • Figs. 6 shows microscopic images of the exobubbles of the invention at 4x and 40x magnification.
  • Figs. 7a & 7b shows microscopic images of the exobubbles in the (a) absence or (b) presence of ultrasound.
  • Figs. 9a & 9b show ultrasound images observed by the Vevo3100 system before and after bubble-popping mode.
  • Figs. 10a - 1 Od show the characterizations of CAR-T cell-derived exobubbles made as described in Example 8.
  • Fig. 11 provides images showing in vitro time-lapse images of the CAR-T cell-derived exobubble treated murine pancreatic cancer cells.
  • 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.
  • 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.
  • incident light easily passes through the particles, resulting in weak scattering intensity. This makes observation challenging in a general optical setting.
  • d Schematic of eVISON preparation 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.
  • Figs.14a -14h provide graphs and images showing optical and physical characterization of eVISON.
  • 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 geometric size distribution of the particles was measured using an electrical sensing technique, h, The schematic represents eVISION's diameters.
  • 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 Alexa Fluor
  • 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.
  • PE Phycoerythrin
  • 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).
  • 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.
  • Exobubbles exhibit unique features: growth dynamic property, high internal pressure, echogenic signal, and a 19 F magnetic resonance (MR) signal.
  • Exobubbles can be used for ultrasound imaging and 19 F 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.
  • 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.
  • a mammal e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig
  • the term does not denote a particular age. Thus, adult, juvenile, and newborn subjects are intended to be covered.
  • 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.
  • treatment refers 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 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.
  • terapéuticaally 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.
  • 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.
  • 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.
  • Extracellular vesicles 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).
  • 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 are highly stable because carbon- fluorine bonds are very strong, and have a high density; over twice that of water.
  • perfluorcarbons examples 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.
  • 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.
  • the perfluorocarbon is encapsulated by the exosome.
  • the interior of the exobubble comprises perfluorocarbon
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Therapeutic agents can also be included in the exobubbles.
  • therapeutic agents include antimicrobial agents, anti-inflammatory agents, immune agents, and anticancer agents.
  • the exobubble further comprises an anticancer agent.
  • 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.
  • the mammalian cell is a T cell, a macrophages, an NK cell, a stem cell, or genetically-engineered cell.
  • 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, BiotiumTM) Immunoprecipitation, Multiplexed ExoSearch Chip, and Acoustic Nanofilter. Doyle L. and Wang M., Cells, 8(7): 727 (2019).
  • the extracellular vesicle and/or exosome is isolated from a cell using filtration, chromatography, precipitation, centrifugation, or a combination of thereof.
  • exosomes can be isolated from HEK293T cells using exoEasy Maxi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.
  • 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) and C10F20O5 (perfluoro-15-crown-5-ether).
  • the perfluorocarbon is selected from C5F12 (perfluoropentane), CeFi4 (perfluorohexane), CsFnBr (perfluorooctyl bromide), C10F20O5 (perfluoro-15-crown-5-ether), or a combination of thereof.
  • 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.
  • PFH 10 - 100 pL
  • exosome solution 1 x 10 9 - 1 x 10 11 particles/mL
  • sonication bath-type or probe-type sonicator, 1 min
  • 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.
  • 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.
  • the subject being treated has been diagnosed with cancer.
  • 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.
  • 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 can invade and destroy nearby tissue and spread to other parts of the body through a process referred to as metastasis.
  • 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.
  • the cancer is selected from the group of cancer types consisting of sarcoma, carcinoma, and lymphoma.
  • 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.
  • 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.
  • 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.
  • DAMPs damage-associated molecular patterns
  • 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).
  • 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.
  • CAR chimeric antigen receptor
  • 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.
  • 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.
  • the exobubble further comprises an anticancer agent.
  • an anticancer agent A wide variety of anticancer agents are known to those skilled in the art.
  • the anticancer agent is a hydrophobic anticancer agent that can easily associate with the exobubble.
  • 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
  • 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.
  • 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.
  • Exobubbles can be used as contrast agents for imaging methods such as ultrasound and 1 9 F 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).
  • MR magnetic resonance
  • 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.
  • CAR chimeric antigen receptor
  • 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.
  • the exobubbles used in the method of imaging can be any of the exobubbles described herein.
  • 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.
  • 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
  • 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 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.
  • 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.
  • the exobubbles are visualized using fluorescent imaging.
  • optical imaging include coherent optical imaging, bright-field microscopy, dark-field microscopy, phase-contrast microscopy, and holography.
  • 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.
  • 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.
  • EVs Circulating extracellular vesicles
  • tiny membrane-bound particles (average diameter; 150 nm) released by cells, are gaining traction as a promising diagnostic tool in clinical settings.
  • biomolecules e.g., proteins, nucleic acids, lipids
  • investigating EVs offers the potential for early disease diagnosis and valuable insights for monitoring disease progression and therapeutic response.
  • 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.
  • extracellular vesicle droplets with optical manipulation 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.
  • PFC perfluorocarbon
  • CeFi4 perfluorohexane, PFH
  • PFH perfluorohexane
  • 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.
  • Qsc cross-sections for scattering
  • eVISION detected a significantly 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.
  • eVISION 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 10 4 objects/mL (bare EV) and 9.62 x 10 6 objects/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).
  • eVISION provides the flexibility to choose fluorescence dye from various libraries for the detection of EVs (Fig. 15g).
  • eVISION In evaluating the performance of eVISION, we harnessed its ultrasensitive capabilities in fluorescence systems to assess its potential for multiplexing various biomolecules in EVs.
  • 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).
  • CD63-GFP + EV 73-fold
  • MemGlow + EV MemGlow + EV (10-fold)
  • Pyronin Y + EV 317-fold
  • Hoechst + EV 8-fold
  • SYBR gold + EVs i.e., EVs containing nucleic acids
  • Cy5 + EVs containing proteins
  • Fig. 16d shows the ultrasensitive triple multiplexing capability of eVISION in a chip.
  • eVISION G1 i 36 EGFR vUI cell-derived
  • APTES 3- aminopropyl)triethoxysilane
  • 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 and accurate detection.
  • 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.
  • 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.
  • ddPCR droplet digital polymerase chain reaction
  • 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.
  • a human glioma cell line expressing epidermal growth factor receptor variant III (Gli36 R ' 7A ' l /// ; RRID: CVCL_RL88) and a human embryonic kidney cell line expressing green fluorescent protein (HEK293T 67A+ ) 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).
  • DMEM Dulbecco’s modified eagle medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • Carlsbad, CA, USA penicillin/streptomycin solution
  • 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.
  • NTA Nanoparticle tracking analysis
  • 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.
  • HT-X1 high-performance holotomography microscope
  • 10 pL of eVISION to a 35 mm glass bottom dish and covered it with a glass coverslip.
  • HT-X1 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).
  • eVISION using the “pre-mixing strategy” or the “after-mixing strategy.”
  • 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.
  • 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).
  • eVISION 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 the ultrasound 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.
  • FDTD Finite-difference time-domain 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.
  • 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.

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Abstract

Des exo-bulles comprenant un perfluorocarbone encapsulé par une vésicule extracellulaire et/ou un exosome sont décrites. Des méthodes de préparation des exo-bulles, des méthodes de traitement du cancer à l'aide des exo-bulles, et des méthodes d'imagerie à l'aide des exo-bulles.
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WO2020047071A1 (fr) * 2018-08-31 2020-03-05 Timothy Bertram Compositions comprenant des vésicules d'origine cellulaire et utilisations associées
EP1928306B1 (fr) * 2005-09-29 2021-01-13 General Hospital Corporation Systèmes et procedes de tomographie par cohérence optique incluant une imagerie microscopique par fluorescence d'une ou plusieurs structures biologiques
US20230058977A1 (en) * 2020-11-03 2023-02-23 Vesselon, Inc. Compositions and methods for targeted delivery of therapeutics using carriers

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Publication number Priority date Publication date Assignee Title
EP1928306B1 (fr) * 2005-09-29 2021-01-13 General Hospital Corporation Systèmes et procedes de tomographie par cohérence optique incluant une imagerie microscopique par fluorescence d'une ou plusieurs structures biologiques
WO2020047071A1 (fr) * 2018-08-31 2020-03-05 Timothy Bertram Compositions comprenant des vésicules d'origine cellulaire et utilisations associées
US20230058977A1 (en) * 2020-11-03 2023-02-23 Vesselon, Inc. Compositions and methods for targeted delivery of therapeutics using carriers

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