WO2019023584A1 – January 31, 2019 – FIXATION AND RETENTION OF EXTRACELLULAR VESICLES

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Inventors :

PENA, John, T. G. - 679 St. Marks Avenue, Apt. 2, Brooklyn, NY 11216

Owner :

CORNELL UNIVERSITY

Application Number :

WOUS18044102

Document Number :

WO2019023584A1

Priority Date :

January 1, 1970

Filing Date :

July 27, 2018

Date of Grant/ Publication :

January 31, 2019

Class :

G01N21 / 75; G01N33 / 48

Abstract

The present invention relates to a method of fixing extracellular vesicles. The method includes providing a sample containing extracellular vesicles and contacting the sample with a non-reversible cross-linking agent and, optionally, an aldehyde-containing fixative. Preferably the non-reversible cross-linking agent is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The method also includes imaging the fixed extracellular vesicles for the determination or exclusion of disease or disorder in a clinical sample. The present invention also relates to a kit for fixing extracellular vesicles in a biological sample.

Claim(s)

1. A method of fixing extracellular vesicles, said method comprising: providing a sample containing extracellular vesicles and contacting the sample with a non-reversible cross-linking agent under conditions effective to fix the extracellular vesicles.;
2. The method of claim 1 further comprising: contacting said sample with an aldehyde-containing fixative before, after, or at the same time as said contacting the sample with a non-reversible cross-linking agent to fix the extracellular vesicles.;
3. The method of claim 2, wherein said extracellular vesicles are selected from the group consisting of exomeres, exosomes, multivesicular bodies, intraluminal vesicles (ILVs), multivesicular endosomes (MVEs), oncosomes, micro-vesicles ranging in size from 20-10,000 nm, apoptotic bodies, and vesicles originating from endosome or plasma membranes.;
4. The method of claim 2, wherein the sample is a biological fluid or tissue.;
5. The method of claim 4, wherein the sample is a biological fluid selected from the group consisting of blood products, sols, suspensions, gels, colloids, fluids, liquids, plasmas, plastic solids, suspension, gels, breast milk, nipple aspirate fluid, urine, semen, amniotic fluid, cerebrospinal fluid, vitreous, aqueous humor, synovial fluid, lymph, bile, saliva, bile, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, sebum (skin oil), serous fluid, smegma, sputum, sweat, tears, vaginal secretion, surgical waste, and vomit.;
6. The method of claim 5, wherein the biological fluid sample is vitreous or aqueous humor.;
7. The method of claim 5, wherein the biological fluid sample is a blood product selected from the group consisting of whole blood, blood plasma, blood platelets, and blood serum.;
8. The method of claim 5, wherein the biological fluid sample is urine.;
9. The method of claim 5, wherein the biological fluid sample is cerebrospinal fluid.;
10. The method of claim 5, wherein the biological fluid sample is nipple aspirate fluid. 11. The method of claim 4, wherein the sample is a tissue selected from the group consisting of skin, bone, cartilage, tendon, ligament, vertebral disc, cornea, lens, meniscus, hair, striated muscle, smooth muscle, cardiac muscle, adipose tissue, fibrous tissue, neural tissue, connective tissue, cochlea, testis, ovary, stomach, lung, heart, liver, pancreas, kidney, intestine, and eye. 12. The method of claim 2, wherein the non-reversible cross-linking agent is selected from the group consisting of a water-soluble carbodiimide, cyanogen halide, and mixtures thereof.;
13. The method of claim 12, wherein the non-reversible cross-linking agent is l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide.;
14. The method of claim 12, wherein the non-reversible cross-linking agent is a cyanogen halide selected from the group consisting of cyanogen bromide, cyanogen fluoride, cyanogen chloride, and cyanogen iodide. 15. The method of claim 2 further comprising: contacting the sample with a further cross-linking agent, independently of, and before, after, or at the same time as said contacting with said non-reversible cross-linking agent and as said contacting with said aldehyde-containing fixative, said further cross-linking agent being selected from the group consisting of ethylene glycol di(meth)acrylate, ethylene glycol diacrylate, di(ethylene glycol) diacrylate, tetra(ethylene glycol) diacrylate, ethylene glycol dimethacrylate, di(ethylene glycol) dimethacrylate, tri(ethylene glycol) dimethacrylate, derivatives of methyl enebisacrylamide, N,N- methylenebisacrylamide, N,N- methylenebisacrylamide, N,N- (l,2-dihydroxyethylene)bisacrylamide, formaldehyde-free cross- linking agents, N- (l-hydroxy-2,2-dimethoxyethyl)acrylamide, divinylbenzene, formalin fixatives, formal calcium, formal saline, zinc formalin (unbuffered), Zenker’s fixative, Helly’s fixative, B-5 fixative, Bouin’s solution, Hollande’s solution, Gendre’s solution, Clarke’s solution, Carnoy’s solution, methacarn, alcoholic formalin, and formol acetic alcohol.;
16. The method of claim 2, wherein said extracellular vesicles have a size of 20 nanometers to 10,000 nm.;
17. The method of claim 2 further comprising: imaging the fixed extracellular vesicles.;
18. The method of claim 17, wherein said imaging is carried out by transmission electron microscopy, scanning electron microscopy, cryoelectron microscopy, binocular stereoscopic microscopy, wide-field microscopy, polarizing microscopy, phase contrast microscopy, multi- photon microscopy, differential interference contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, ray microscopy, ultrasonic microscopy, color metric assay, chemiluminescence assay, spectrophotometry, positron emission tomography, computerized tomography, and magnetic resonance imaging. 19. The method of claim 17 further comprising: detecting the extracellular vesicles in the biological sample based on said imaging. 20. The method of claim 19, wherein the biological sample is a clinical sample. 21. The method of claim 20, wherein the clinical sample is from a patient treated with a clinical drug.;
22. The method of claim 17 further comprising: diagnosing whether the subject providing the clinical sample has a disease or disorder based on said imaging.;
23. The method claim 22, wherein the disease or disorder is selected from the group consisting of cancer, inflammatory diseases, infections, degenerative diseases, diseases caused by pathogens, neurological diseases and disorders, and internal dysfunctions.;
24. The method of claim 23, wherein the disease or disorder is an internal dysfunction selected from the group consisting of glaucoma and other ocular diseases.;
25. The method of claim 23, wherein the disease or disorder is an internal dysfunction characterized by an immunodeficiency or hypersensitivity.;
26. The method of claim 25, wherein the immunodeficiency or hypersensitivity is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma, Crohn’s disease, ulcerative colitis, allergic conditions, eczema, asthma, lupus erythematosus (SLE), multiple sclerosis, allergic encephalomyelitis, sarcoidosis, granulomatosis (including Wegener’s granulomatosis), agranulocytosis, vasculitis (including ANCA), aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia, pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, multiple organ injury syndrome, mysathenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman’s Syndrome, Goodpasture’s Syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud’s Syndrome, Sjorgen’s Syndrome, Stevens- Johnson Syndrome, solid organ transplant rejection, graft versus host disease (GVHD), pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies, Reiter’s disease, and Guillain-Barre’ Syndrome. 27. The method of claim 23, wherein the disease or disorder is a cancer selected from the group consisting of acute granulocytic leukemia, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), adenocarcinoma, adenosarcoma, adrenal cancer, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-Cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, bowel cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumors (GIST), germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme (GBM), glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin’s disease, hypopharyngeal cancer, infiltrating ductal carcinoma (IDC), infiltrating lobular carcinoma (LLC), inflammatory breast cancer (IBC), intestinal cancer, intrahepatic bile duct cancer, invasive/infiltrating breast cancer, islet cell cancer, jaw cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastases, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymous, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed gliomas, mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplasia syndrome, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors (NETs), Non-Hodgkin lymphoma (NHL), non-small cell lung cancer, oat cell cancer, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian primary peritoneal carcinoma, ovarian sex cord stromal tumor, Paget’s disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary tumors, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sarcoma (bone), Sarcoma (soft tissue), Sarcoma (uterine), sinus cancer, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cancer, spinal column cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma/thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell cancer (bladder), Transitional cell cancer (kidney), Transitional cell cancer (ovarian), triple-negative breast cancer, tubal cancer, tubular carcinoma, undiagnosed cancer, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.;
claim 21: 28. The method of claim 27, wherein the cancer is ocular cancer.;
claim 22: 29. The method of claim 23, wherein the disease or disorder is a neurological disease selected from the group consisting of Demyleinating Diseases, Multiple Sclerosis, Parkinson’s disease, Huntington’s disease, Creutzfeld- Jakob disease, Alzheimer’s disease, Wilson’s Disease, Spinal muscular atrophy, Lewy body disease, Friedreich’s Ataxia, Autism, and Autism spectrum disorders, synaptic density associated with disease, and Amyotrophic lateral sclerosis (ALS).;
claim 23: 30. The method of claim 23, wherein the disease or disorder is a neurological disorder selected from substance abuse-related disorders, alcohol use disorders, amphetamine-use disorders, cannabis-use disorders, caffeine-induced disorders, cocaine-use disorders, inhalant-use disorders, opioid-use disorders, hallucinogen disorders, sedative-use, hypnotic-use, or anxiolytic- use disorders, polysubstance-use disorders, sexual dysfunctions, sexual arousal disorder, male erectile disorder, male hypoactive disorder, female hypoactive disorder, eating disorders, overeating disorder, bulimia nervosa, anorexia nervosa, anxiety, obsessive compulsive disorder syndromes, panic attacks, post-traumatic stress disorder, agoraphobia, obsessive and compulsive behavior, impulse control disorders, pathological gambling, intermittent explosive disorder, kleptomania, pyromania, personality disorders, schizoid personality disorder, paranoid personality disorder, schizotypal personality disorder, borderline personality disorder, narcissistic personality disorder, histrionic personality disorder, obsessive compulsive personality disorder, avoidant personality disorder, dependent personality disorder, and antisocial personality disorder, schizophrenia subtypes, schizoaffective disorder, schizophrenia undifferentiated, delusional disorder, cyclothymic disorder, somatoform disorder, hypochondriasis, dissociative disorder, and depersonalization disorder.;
claim 24: 31. The method of claim 23, wherein the disease is a cardiovascular disease.;
claim 25: 32. The method of claim 22, wherein said diagnosing comprises performing two or more assays for disease markers.;
claim 26: 33. The method of claim 22, wherein said diagnosing comprises: providing a standard image of a clinical sample containing extracellular vesicles fixed with the non-reversible cross-linking agent, from a subject having a particular disease or disorder; comparing the image of the clinical sample of the subject to the standard image with regard to size, density, morphology, or spacial distribution of the fixed extracellular vesicles; and determining if the subject has a disease or disorder based on said comparing.;
claim 27: 34. The method of claim 33, wherein the standard image of the clinical sample containing extracellular vesicles is fixed with an aldehyde-containing fixative before, after, or at the same time as it is fixed with the non-reversible cross-linking agent. 35. The method of claim 33 further comprising: administering a therapeutic agent to the subject based on said determining.;
claim 28: 36. The method of claim 22, wherein said diagnosing involves monitoring progression or regression of a particular disease or disorder, providing a prior image of a clinical sample of a subject, containing extracellular vesicles fixed with a non-reversible cross-linking agent; comparing the image of the clinical sample of said subject containing the extracellular vesicles fixed with the non-reversible cross-linking agent to the prior image with regard to size, density, morphology, or spacial distribution of the fixed extracellular vesicles; and determining if the particular disease or disorder is progressing or regressing based on said comparing.;
claim 29: 37. The method of claim 36, wherein the clinical samples containing extracellular vesicles are fixed with an aldehyde-containing fixative before, after, or at the same time as they are fixed with the non-reversible cross-linking agent.;
claim 30: 38. The method of claim 36 further comprising: administering a therapeutic agent to the subject based on said determining. 39. A kit for fixing extracellular vesicles in a biological sample, said kit comprising: a support substrate for holding the sample; and a non-reversible cross-linking agent. The kit according to claim 39 further compri an aldehyde-containing fixative. 41. The kit according to claim 39, wherein the non-reversible cross-linking agent is selected from the group consisting of a water-soluble carbodiimide, cyanogen halide, and mixtures thereof. 42. The kit of claim 41, wherein the non-reversible cross-linking agent is l-ethyl-3-(3- dimethylaminopropyl) carbodiimide.

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