WO2021035135A1 - High-brightness fluorophores for quantification and phenotyping of extracellular vesicles - Google Patents
High-brightness fluorophores for quantification and phenotyping of extracellular vesicles Download PDFInfo
- Publication number
- WO2021035135A1 WO2021035135A1 PCT/US2020/047378 US2020047378W WO2021035135A1 WO 2021035135 A1 WO2021035135 A1 WO 2021035135A1 US 2020047378 W US2020047378 W US 2020047378W WO 2021035135 A1 WO2021035135 A1 WO 2021035135A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- linker
- carrier
- compound
- nanomaterial
- biomolecule
- Prior art date
Links
- 238000011002 quantification Methods 0.000 title description 2
- 239000002086 nanomaterial Substances 0.000 claims abstract description 40
- 150000001875 compounds Chemical class 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 17
- 230000004069 differentiation Effects 0.000 claims abstract description 6
- PZNSFCLAULLKQX-UHFFFAOYSA-N Boron nitride Chemical group N#B PZNSFCLAULLKQX-UHFFFAOYSA-N 0.000 claims description 33
- 239000002202 Polyethylene glycol Substances 0.000 claims description 30
- 229920001223 polyethylene glycol Polymers 0.000 claims description 30
- 125000000524 functional group Chemical group 0.000 claims description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 17
- 239000002041 carbon nanotube Substances 0.000 claims description 15
- 229910021393 carbon nanotube Inorganic materials 0.000 claims description 13
- 230000002209 hydrophobic effect Effects 0.000 claims description 13
- 229910052582 BN Inorganic materials 0.000 claims description 11
- 238000000149 argon plasma sintering Methods 0.000 claims description 10
- 239000002071 nanotube Substances 0.000 claims description 10
- 239000002096 quantum dot Substances 0.000 claims description 5
- 239000000969 carrier Substances 0.000 description 16
- 238000001514 detection method Methods 0.000 description 11
- 239000000090 biomarker Substances 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 239000000104 diagnostic biomarker Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 150000003904 phospholipids Chemical class 0.000 description 3
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical compound C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 229910021389 graphene Inorganic materials 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002356 laser light scattering Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002135 nanosheet Substances 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- -1 polyvinylpyrolidone Polymers 0.000 description 2
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000004611 spectroscopical analysis Methods 0.000 description 2
- 229920003169 water-soluble polymer Polymers 0.000 description 2
- LVNGJLRDBYCPGB-LDLOPFEMSA-N (R)-1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-LDLOPFEMSA-N 0.000 description 1
- SLLFVLKNXABYGI-UHFFFAOYSA-N 1,2,3-benzoxadiazole Chemical class C1=CC=C2ON=NC2=C1 SLLFVLKNXABYGI-UHFFFAOYSA-N 0.000 description 1
- GUXJXWKCUUWCLX-UHFFFAOYSA-N 2-methyl-2-oxazoline Chemical compound CC1=NCCO1 GUXJXWKCUUWCLX-UHFFFAOYSA-N 0.000 description 1
- GJCOSYZMQJWQCA-UHFFFAOYSA-N 9H-xanthene Chemical compound C1=CC=C2CC3=CC=CC=C3OC2=C1 GJCOSYZMQJWQCA-UHFFFAOYSA-N 0.000 description 1
- 102100025222 CD63 antigen Human genes 0.000 description 1
- 102100027221 CD81 antigen Human genes 0.000 description 1
- 102100037904 CD9 antigen Human genes 0.000 description 1
- 101100347633 Drosophila melanogaster Mhc gene Proteins 0.000 description 1
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 1
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 1
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- SUHOOTKUPISOBE-UHFFFAOYSA-N O-phosphoethanolamine Chemical compound NCCOP(O)(O)=O SUHOOTKUPISOBE-UHFFFAOYSA-N 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 150000001251 acridines Chemical class 0.000 description 1
- FZEYVTFCMJSGMP-UHFFFAOYSA-N acridone Chemical class C1=CC=C2C(=O)C3=CC=CC=C3NC2=C1 FZEYVTFCMJSGMP-UHFFFAOYSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- FZICDBOJOMQACG-UHFFFAOYSA-N benzo[h]isoquinoline Chemical compound C1=NC=C2C3=CC=CC=C3C=CC2=C1 FZICDBOJOMQACG-UHFFFAOYSA-N 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002583 cell-derived microparticle Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 150000004775 coumarins Chemical class 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol Substances OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 229930004094 glycosylphosphatidylinositol Natural products 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- QDLAGTHXVHQKRE-UHFFFAOYSA-N lichenxanthone Natural products COC1=CC(O)=C2C(=O)C3=C(C)C=C(OC)C=C3OC2=C1 QDLAGTHXVHQKRE-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 150000003019 phosphosphingolipids Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- ZWZWYGMENQVNFU-UHNVWZDZSA-N sn-glycero-3-phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(=O)OC[C@H](O)CO ZWZWYGMENQVNFU-UHNVWZDZSA-N 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical class [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1486—Counting the particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70539—MHC-molecules, e.g. HLA-molecules
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
Definitions
- PCT/US2020/035568 filed June 1, 2020
- International Application No. PCT/US2020/035574 filed June 1, 2020.
- U.S. Provisional Application Serial No. 62/889,691 International Application No. PCT/US2020/035568
- International Application No. PCT/US2020/035574 are hereby incorporated by reference herein in their entireties.
- Fluorophores are compounds with fluorescent properties that have biomedical applications.
- fluorophores can be used as tracers or dyes for staining certain molecules or structures. More particularly, fluorophores can be used to stain tissues, cells, or biological materials in a variety of analytical methods, such as fluorescent imaging and spectroscopy.
- Extracellular vesicles are biological particles encapsulated with a phospholipid bilayer. EVs have diameter around 20 nanometers (nm) to a few microns but mostly are smaller than around 350 nm. Detection of EVs can be helpful in certain clinical applications such as early disease detection and treatment by diagnostic biomarkers and therapeutics. However, due to their small diameter, EVs only have a few biological markers. Therefore, it is difficult to accurately count and to phenotype small EVs. High-resolution imaging flow cytometry is the most promising method for counting EVs by laser light scattering and phenotyping them by fluorescent signals of fluorophores that tagged on them.
- a compound according to an exemplary embodiment of this disclosure, among other possible things includes a nanomaterial carrier, a first linker having a first end connected to the nanomaterial carrier, a second linker having a second end connected to the nanomaterial carrier, a fluorescent entity connected to a second end of the first linker, and a biomolecule connected to a second end of the second linker.
- the biomolecule is configured to connect to a cluster of differentiation (CD) of an extracellular vesicle (EV).
- the nanomaterial carrier is a boron nitride nanotube (BNNT) or carbon nanotube (CNT).
- the nanomaterial is a nanodot.
- the first end of at least one of the first and second linkers is covalently bonded to the nanomaterial carrier.
- the first end of at least one of the first and second linkers includes a functional group, and the functional group covalently bonds the linker to the nanomaterial carrier.
- the second end of at least one of the first second linkers ins covalently bonded to the fluorescent entity or the biomolecule via a functional group.
- the first and of at least one of the first and second linkers is non-covalently bonded to the nanomaterial carrier.
- At least one of the first and second linkers is amphiphilic, and includes a hydrophobic region and a hydrophilic region.
- the hydrophobic region is non-covalently bonded to the nanomaterial carrier.
- the linkers has a molecular weight between about 1000 and 10000 Da.
- the nanomaterial carrier is a boron nitride nanotube.
- At least one of the first and second linkers is DSPE-PEGn (l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[ (polyethylene glycol)n]), where n is a number of polyethylene glycol (PEG) molecules in a PEG chain.
- a method includes linking at least one fluorescent entity and at least one biomolecule to a nanomaterial carrier.
- the biomolecule is configured to connect a cluster of differentiation (CD) of an extracellular vesicle (EV) to form a compound.
- the method also includes applying the compound to an EV such that the compound connects to the EV via the biomolecule to form a marked EV, and detecting at least one of light scattering and fluorescence of the marked EV.
- the carrier is a boron nitride nanotube (BNNT) carrier, a carbon nanotube (CNT) carrier, or a nanodot.
- BNNT boron nitride nanotube
- CNT carbon nanotube
- the linking of at least one of the fluorescent entity and the biomolecule is via a linker.
- the linking of the linker to the nanomaterial carrier is via a covalent bond.
- a first end of the linker includes a first functional group and a second end of the linker includes a second functional group.
- the first functional group covalently bonds to the nanomaterial carrier and the second functional group covalently bonds to the fluorescent entity.
- the linking of at least one of the fluorescent entity and the biomolecule is via a linker.
- the linking of the linker to the nanomaterial carrier is via a non-covalent bond.
- the linker is amphiphilic, and includes a hydrophobic region and a hydrophilic region.
- the hydrophobic region is non-covalently bonded to the nanomaterial carrier.
- the linker has a molecular weight between about 1000 and 10000 Da.
- the nanomaterial carrier is a boron nitride nanotube.
- the linker is DSPE- PEGn ( 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ (polyethylene glycol)n]), where n is a number of polyethylene glycol (PEG) molecules in a PEG chain.
- FIG 1A schematically shows high-brightness fluorophore structures.
- FIG IB schematically shows dye-linker structures.
- FIG 1C schematically shows antibody-linker structures.
- FIG 2 A shows light scattering signals from a PBS buffer solution including EVs.
- FIG 2B shows fluorescence signals from the PBS buffer solution of Figure 2A.
- FIG 3A shows light scattering signals from short BNNTs.
- FIG 3B shows light scattering signals from short BNNTs at 4X BNNT concentration.
- FIG 3C shows the length distribution of the short BNNTs.
- FIG 4A shows light scattering signals from short BNNTs labeled with dye-linkers.
- FIG 4B shows fluorescence signals from short BNNTs labeled with dye-linkers.
- FIG 5A shows light scattering signals from long BNNTs.
- FIG 5B shows the length distribution of the long BNNTs.
- high-brightness fluorophores contain a carrier element, a fluorescent element, and a linker linking the carrier element to the fluorescent element.
- a carrier element for biomedical applications, each of the carrier element, the linker, and the fluorescent element must be biocompatible (though the requirements for biocompatibility will vary with the particular application).
- One example carrier element is a nanomaterial, such as carbon nanotubes (CNT) and boron nitride nanotubes (BNNTs), both of which are recognized as biologically compatible nanomaterials for biomedical applications such as cellular drug delivery and spectroscopy applications.
- CNT carbon nanotubes
- BNNTs boron nitride nanotubes
- fluorescent elements linked to nanotubes exhibit quenching, or a reduction in the brightness of the fluorescence.
- Extracellular vesicles are biological particles encapsulated with a phospholipid bilayer. EVs are naturally released biological particles from cells but cannot replicate by themselves. EVs have diameter around 20 nanometers (nm) to a few microns but mostly are smaller than around 350 nm. A wide variety of EV subtypes have been proposed as defined by their size, cellular source, and function, including exosomes ( ⁇ 20-150nm), ectosomes ( ⁇ 150-1000nm) and apoptotic bodies ( ⁇ l-5 um). EVs can be found in biological fluids including blood, urine, and cerebrospinal fluid. They also release into the growth medium of cultured cells.
- EVs carry various proteins, nucleic acids, metabolites, and even organelles from their parent cells.
- EVs carry some biomarkers / biological molecules on their surfaces, the so-called cluster of differentiation (CD).
- CDs cluster of differentiation
- Fluorophores 20 are schematically shown.
- Fluorophores 20 generally comprise an inorganic nano-scale carrier 22, a linker 24, a fluorescent entity 26, as well as one or more biomolecules 28 (such as antibodies).
- the biomolecules 28 can be selected to interact with biomarkers on EVs.
- Example biomarkers include surface markers such as MHC, CD9, CD63, CD81 etc. This interaction connects the fluorophore 20 to the EV by interaction between the biomarker 28 on the fluorophore and the CD(s) so that the EV. In this way, the EV can be detected (and counted, identified, etc.) by detection of the fluorophore 20, as discussed in more detail below.
- the carrier 22 is, in one example, a BNNT or CNT carrier.
- the carrier 22 can be fabricated by any known method.
- the carrier 22 is a multi- walled BNNT or CNT carrier, where each BNNT or CNT has multiple co-axial shells of hexagonal boron nitride (h-BN for BNNTs) or graphene (for CNTs), with a typical external diameter of more than about 1 nm but less than about 80 nm.
- the length of these BNNTs and CNTs is between about 1-5000 nm.
- the carrier 22 can be another nano scale inorganic material, such as boron nitride (h-BN) nanosheets/nanoparticles and graphene/graphite nanosheets/nanoparticles.
- the nanodots are processed by mechanical agitation to encourage the formation of imperfections in the nanostructure of the dots, which imperfections encourage/enable bonding to linkers 24, which in turn enables more linkers 24 and thus more fluorescent entities 26 to bond to the nanodot and improve fluorescence of the resulting fluorophore 20.
- the linker 24 is an amphiphilic polymeric linker. That is, the linker 24 includes a hydrophobic region 25 and a hydrophilic region 27. The hydrophobic region 25 non-covalently bonds to the nanotube carrier 22, while the hydrophilic region 27 is covalently bonded to the fluorescent entity 26 (or another entity, as will be discussed below).
- One example linker 24 is DSPE-PEG n (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ (polyethylene glycol) n ]), where n is a number of polyethylene glycol (PEG) molecules in a PEG chain.
- Other linkers 24 can similarly include a PEG chain (or a different chain) which varies in length.
- the hydrophilic region 27 is covalently bonded to the biomolecule 28 (such as an antibody, nucleic acid, etc).
- One example linker 24 is DSPE-PEG n (1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[ (polyethylene glycol) n ]), where n is a number of polyethylene glycol (PEG) molecules in a PEG chain.
- Other linkers 24 can similarly include a PEG chain (or a different chain) which varies in length.
- the liker 24 has a molecular weight of greater than about 1000 Da (which corresponds to a stretched linker length of about 5-10 nm for a linker 24 with a PEG chain) and less than about 10000 Da, which allows for improved fluorescence of the resulting fluorophores 20 as compared to prior art fluorophores.
- the linker 24 molecular weight is greater than about 2400 Da and less than about 10000 Da.
- a linker comprising more than one of the above groups will be selected such that the linker 24 is stable (e.g., not prone to degradation) and biologically appropriate; for example, a linker 24 may not include two adjacent -O- groups, which would generate an unstable peroxide linkage.
- the linker 24 may be a straight chain, a branched chain, or may include one or more ring systems.
- Non-limiting exemplary linkers include a hydrophobic area which can be fatty acids, phospholipids, sphingolipids, phosphosphingolipids [such as DSPE, l-0-hexadecanyl-2-0-(9Z-octadecenyl)-sn- glycero-3-phospho-(l'-rac-glycerol) (ammonium salt), N-octanoyl-sphingosine-1- ⁇ succinylfmethoxy (poly ethylene glycol)5000, D-erythro-sphingosyl phosphoethanolamine, l,2-diphytanoyl-sn-glycero-3-phospho-L-serine, 3-sn- phosphatidyl-L-serine (PS), glycosylphosphatidylinositol, l,2-dioleoyl-sn-glycero-3- phosphoethanoamine but not limited).
- the hydrophobic unit can be used to conjugate with water soluble polymeric chains such as PEG (or PEO polyethyleneoxide), PMO (poly methyl oxazoline), PEI (polyethyleneimine), polyvinyl alcohol, polyvinylpyrolidone, polyacrylamide, polypeptide, carbohydrate anchors.
- PEG or PEO polyethyleneoxide
- PMO poly methyl oxazoline
- PEI polyethyleneimine
- polyvinyl alcohol polyvinylpyrolidone
- polyacrylamide polypeptide
- carbohydrate anchors e.g., ethyleneimine
- the watersoluble polymeric chains are attached to the linkers at one end, and attached to the fluorescent entity (or another moiety, as discussed below) at a second end.
- These hydrophobic and hydrophilic units must have reactive groups as mentioned above and such that the groups conjugate together into amphiphilic linkers.
- the fluorescent entity 26 is any know fluorescent dye, including but not limited to coumarins, benzoxadiazoles, acridones, acridines, bisbenzimides, indole, benzoisoquinoline, naphthalene, anthracene, xanthene, pyrene, porphyrin, fluorescein, rhodamine, boron-dipyrromethene (BODIPY) and cyanine derivatives. Many such fluorescent dyes are commercially available.
- the fluorescent entity 26 is bonded to the linker 24 by any appropriate method, such as by inducing a chemical reaction between the linker 24 and fluorescent entity 26, as is known in the art.
- fluorophores 20 can be created by covalent functionalization of the linkers 24 onto the carrier 22.
- the linker 24 includes a functional group “R” that interacts with the carrier 22 and a functional group “R”’ that interacts with other moieties that are attached to the carrier 22, like the fluorescent dye molecules 26 and the antibodies 28.
- An example functional group is a hydroxyl group, though any known functional group is contemplated.
- the carriers 22 are processed such as by mechanical agitation in polar liquid in order to form imperfections in the nanostructure of the carriers 22, which imperfections encourage/enable bonding to linkers 24 via functional groups R.
- BNNT carriers 22 before conjugation with linker 24 and fluorescent entity 26 alone can initiate light scattering sufficient for detection. Therefore, in another example, the BNNT carriers 22 can be used as the carriers of fluorophores 26 without linkers 24 for the detection of EVs through flow cytometry measurements.
- FIG 2A shows the forward scattering (FSC) and side scattering (SSC) of laser lights of the Phosphate-buffered saline (PBS) solution that includes EVs.
- FSC of 10 3 are initiated by laser with a wavelength of 405 nm
- SSC of 10 2 are initiated by laser with a longer wavelength of 488 nm.
- These levels of scattering signals are considered as “noise” for prior art methods of EV detection. Therefore, there is a detection window R1 with higher scattering strength for nano particles, which would include EVs as well as the carriers 22 of the fluorophores 20.
- the flow rate for all measurements discussed herein was 0.25pl/s and the laser powers are lOOmW for both 405nm and 488nm.
- FIG 2B shows the fluorescence signals collected by the FITC channel (centered around 52 nm) for the PBS solution. As shown, only noise is detected as there are no fluorophores 20 in the sample.
- FIG 3A shows the FSC and SSC of short BNNT carriers 22.
- “short” BNNTs have a length that is less than about 500 nm, with an average length of about 330nm.
- the particle concentration of these BNNTs is 2.5 x 10 8 /ml.
- higher scattering signals are detected within the R1 window defined in Figure 2A.
- the mean signal strength within the window is (2,698, 1,150). This means, BNNTs are detectable by laser light scattering in a flow cytometer and therefore can be quantified. This will help to quantify EVs when stained with fluorophores 20 having BNNT carriers 22 such as those described here.
- FIG 3B shows the FSC and SSC of the BNNT carriers 22 when concentrated by 4X.
- signals are detected within the R1 window, with the mean signal strength of (1,043, 934). This means, the detectable scattering signal depends on the concentration of the BNNTs. In this particular case, some of the strong scatterings are not being recorded at higher particle concentration (1.0 x 10 9 /ml).
- the length distribution of these short BNNTs is shown in FIG 3C.
- FIG 4A shows the FSC and SSC of fluorophores 20 having short BNNT carriers, linkers 24, and a fluorescent entity 26 (in this example, FITC dye). As shown, strong scattering signals are detected within the R1 window, with a mean strength of (2,627, 1,455).
- FIG 4B shows the fluorescence signals of fluorophores 20 having short BNNT carriers, linkers 24, and a FITC entity 26. As shown, a strong signal centered around 10 3 are detected. This means, both the scattering and fluorescence signal of the fluorophores 20 having short BNNT carriers, linkers 24, and a fluorescent entity 26 are also detectable under the conventional settings/circumstances for light scattering and fluorescence detection, and thus suitable for use in EV labeling and detection.
- the BNNT carriers 22 can be “long” BNNTs.
- “long” BNNTs have a length between about 500 and 5000 nm. More particularly, long BNNTs have a length between about 500 and 2000 nm.
- FIG. 5B shows the length distribution of an example “long” BNNT sample, which has a mean length of 900 nm, with many BNNTs longer than 1000 nm.
- FIG 5A shows the FSC and SSC of the long BNNT carriers. The particle concentration of this BNNT sample is 6 xlO 10 /ml. As shown, the strength of the scattering signals strong, with a mean strength within the R1 window of (3,221, 739).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nanotechnology (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Dispersion Chemistry (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2022511205A JP2022544703A (en) | 2019-08-21 | 2020-08-21 | Bright fluorescent dyes for extracellular vesicle quantification and phenotyping |
CN202080074598.2A CN114631016A (en) | 2019-08-21 | 2020-08-21 | High brightness fluorophores for quantification and phenotypic analysis of extracellular vesicles |
CA3148704A CA3148704A1 (en) | 2019-08-21 | 2020-08-21 | High-brightness fluorophores for quantification and phenotyping of extracellular vesicles |
EP20764562.3A EP4018192A1 (en) | 2019-08-21 | 2020-08-21 | High-brightness fluorophores for quantification and phenotyping of extracellular vesicles |
US17/637,018 US20220291207A1 (en) | 2019-08-21 | 2020-08-21 | High-brightness fluorophores for quantification and phenotyping of extracellular vesicles |
KR1020227009150A KR20220057550A (en) | 2019-08-21 | 2020-08-21 | High-brightness fluorophores for quantification and phenotyping of extracellular vesicles |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201962889691P | 2019-08-21 | 2019-08-21 | |
US62/889,691 | 2019-08-21 | ||
PCT/US2020/035574 WO2020243710A1 (en) | 2019-05-31 | 2020-06-01 | High-brightness nanotube fluorophores by covalent functionalization |
PCT/US2020/035568 WO2021002987A1 (en) | 2019-05-31 | 2020-06-01 | High-brightness nanodot fluorophores by covalent functionalization |
USPCT/US2020/035574 | 2020-06-01 | ||
USPCT/US2020/035568 | 2020-06-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021035135A1 true WO2021035135A1 (en) | 2021-02-25 |
Family
ID=74659729
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2020/047378 WO2021035135A1 (en) | 2019-08-21 | 2020-08-21 | High-brightness fluorophores for quantification and phenotyping of extracellular vesicles |
Country Status (7)
Country | Link |
---|---|
US (1) | US20220291207A1 (en) |
EP (1) | EP4018192A1 (en) |
JP (1) | JP2022544703A (en) |
KR (1) | KR20220057550A (en) |
CN (1) | CN114631016A (en) |
CA (1) | CA3148704A1 (en) |
WO (1) | WO2021035135A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20180296705A1 (en) | 2017-04-13 | 2018-10-18 | Michigan Technological University | High-brightness fluorophores |
-
2020
- 2020-08-21 CN CN202080074598.2A patent/CN114631016A/en active Pending
- 2020-08-21 US US17/637,018 patent/US20220291207A1/en active Pending
- 2020-08-21 CA CA3148704A patent/CA3148704A1/en active Pending
- 2020-08-21 KR KR1020227009150A patent/KR20220057550A/en unknown
- 2020-08-21 JP JP2022511205A patent/JP2022544703A/en active Pending
- 2020-08-21 EP EP20764562.3A patent/EP4018192A1/en active Pending
- 2020-08-21 WO PCT/US2020/047378 patent/WO2021035135A1/en unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20180296705A1 (en) | 2017-04-13 | 2018-10-18 | Michigan Technological University | High-brightness fluorophores |
WO2018191690A1 (en) * | 2017-04-13 | 2018-10-18 | Michigan Technological University | High-brightness fluorophores |
Non-Patent Citations (4)
Title |
---|
ANUSH ARAKELYAN ET AL: "Antigenic composition of single nano-sized extracellular blood vesicles", NANOMEDICINE , NANOTECHNOLOGY , BIOLOGY AND MEDICINE, vol. 11, no. 3, April 2015 (2015-04-01), NL, pages 489 - 498, XP055750725, ISSN: 1549-9634, DOI: 10.1016/j.nano.2014.09.020 * |
MAHNOUSH TAYEBI ET AL: "A MoS 2 -MWCNT based fluorometric nanosensor for exosome detection and quantification", NANOSCALE ADVANCES, vol. 1, no. 8, 6 August 2019 (2019-08-06), pages 2866 - 2872, XP055750719, ISSN: 2516-0230, DOI: 10.1039/C9NA00248K * |
SAPSFORD K E ET AL: "Functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology", CHEMICAL REVIEWS, AMERICAN CHEMICAL SOCIETY, US, vol. 113, no. 3, 13 March 2013 (2013-03-13), pages 1904 - 2074, XP002722245, ISSN: 0009-2665, [retrieved on 20130322], DOI: 101021/CR300143V * |
VENDULA POSPICHALOVA ET AL: "Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer", JOURNAL OF EXTRACELLULAR VESICLES, vol. 4, no. 1, 2015, pages 1 - 15, XP055750730, DOI: 10.3402/jev.v4.25530 * |
Also Published As
Publication number | Publication date |
---|---|
CA3148704A1 (en) | 2021-02-25 |
CN114631016A (en) | 2022-06-14 |
US20220291207A1 (en) | 2022-09-15 |
JP2022544703A (en) | 2022-10-20 |
KR20220057550A (en) | 2022-05-09 |
EP4018192A1 (en) | 2022-06-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhang et al. | Nanotechnology in cancer diagnosis: progress, challenges and opportunities | |
Kurian et al. | Elucidating methods for isolation and quantification of exosomes: a review | |
Chen et al. | Aptamer-conjugated nanomaterials for bioanalysis and biotechnology applications | |
Wang et al. | Quantum dots, lighting up the research and development of nanomedicine | |
Montalti et al. | Dye-doped silica nanoparticles as luminescent organized systems for nanomedicine | |
Rollett et al. | Folic acid-functionalized human serum albumin nanocapsules for targeted drug delivery to chronically activated macrophages | |
Gao et al. | In vivo cancer targeting and imaging with semiconductor quantum dots | |
US9797840B2 (en) | Highly fluorescent polymer nanoparticle | |
Zrazhevskiy et al. | Designing multifunctional quantum dots for bioimaging, detection, and drug delivery | |
Mansur et al. | Water-soluble nanoconjugates of quantum dot-chitosan-antibody for in vitro detection of cancer cells based on “enzyme-free” fluoroimmunoassay | |
US20110158901A1 (en) | Chitosan-based nanoparticles and methods for making and using the same | |
Prabhakar et al. | Functionalization of graphene oxide nanostructures improves photoluminescence and facilitates their use as optical probes in preclinical imaging | |
Chen et al. | Aggregation-induced emission luminogen assisted self-assembly and morphology transition of amphiphilic glycopolypeptide with bioimaging application | |
EP2549925B1 (en) | Multimodal diagnostic technology for early stage cancer lesions | |
Chen et al. | Inorganic fluorescent nanoprobes for cellular and subcellular imaging | |
WO2013043902A1 (en) | Targeted nanoparticles joined to reporter molecules through multiple mechanisms | |
Han et al. | Fate of antibody-targeted ultrasmall gold nanoparticles in cancer cells after receptor-mediated uptake | |
Domingo et al. | Preparation of PEG-grafted immunomagnetoliposomes entrapping citrate stabilized magnetite particles and their application in CD34+ cell sorting | |
Ding et al. | Biofunctionalization of nanoparticles for cytosensing and cell surface carbohydrate assay | |
US20220291207A1 (en) | High-brightness fluorophores for quantification and phenotyping of extracellular vesicles | |
Levin et al. | Enhanced cellular internalization of near-infrared fluorescent single-walled carbon nanotubes facilitated by a transfection reagent | |
CN114225044B (en) | Reagent for modifying extracellular vesicles and preparation method thereof | |
Gulati et al. | Nanomedicine: Potential devices for diagnostics | |
WO2021023997A1 (en) | Light emitting marker and assay method | |
Ghazizadeh et al. | The viewpoint of nanolipid vesicles (liposomes, exosomes, and microvesicles) as biosensors in medical health advances |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20764562 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3148704 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2022511205 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20227009150 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2020764562 Country of ref document: EP Effective date: 20220321 |