GB2567124A - Imaging agents and methods - Google Patents
Imaging agents and methods Download PDFInfo
- Publication number
- GB2567124A GB2567124A GB1707342.0A GB201707342A GB2567124A GB 2567124 A GB2567124 A GB 2567124A GB 201707342 A GB201707342 A GB 201707342A GB 2567124 A GB2567124 A GB 2567124A
- Authority
- GB
- United Kingdom
- Prior art keywords
- compound
- formula
- group
- imaging
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 43
- 239000012216 imaging agent Substances 0.000 title description 2
- 150000001875 compounds Chemical class 0.000 claims abstract description 67
- -1 halide anion Chemical class 0.000 claims abstract description 39
- 238000003384 imaging method Methods 0.000 claims abstract description 32
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 18
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 13
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims abstract description 11
- 230000001678 irradiating effect Effects 0.000 claims abstract description 11
- 125000003118 aryl group Chemical group 0.000 claims abstract description 9
- 125000001153 fluoro group Chemical group F* 0.000 claims abstract description 9
- 238000002073 fluorescence micrograph Methods 0.000 claims abstract description 7
- 125000001072 heteroaryl group Chemical group 0.000 claims abstract description 6
- 125000004076 pyridyl group Chemical group 0.000 claims abstract description 6
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims abstract description 6
- 125000000339 4-pyridyl group Chemical group N1=C([H])C([H])=C([*])C([H])=C1[H] 0.000 claims abstract description 4
- 125000002541 furyl group Chemical group 0.000 claims abstract description 4
- 125000005956 isoquinolyl group Chemical group 0.000 claims abstract description 4
- 125000001624 naphthyl group Chemical group 0.000 claims abstract description 4
- 125000005493 quinolyl group Chemical group 0.000 claims abstract description 4
- 125000001544 thienyl group Chemical group 0.000 claims abstract description 4
- 150000001450 anions Chemical class 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 30
- 125000000217 alkyl group Chemical group 0.000 claims description 18
- 210000001519 tissue Anatomy 0.000 claims description 16
- 210000000557 podocyte Anatomy 0.000 claims description 13
- 241001465754 Metazoa Species 0.000 claims description 11
- 229910052739 hydrogen Inorganic materials 0.000 claims description 9
- 239000001257 hydrogen Substances 0.000 claims description 9
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 9
- 210000003463 organelle Anatomy 0.000 claims description 9
- 125000001424 substituent group Chemical group 0.000 claims description 7
- 230000001413 cellular effect Effects 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 125000005010 perfluoroalkyl group Chemical group 0.000 claims description 4
- 125000004211 3,5-difluorophenyl group Chemical group [H]C1=C(F)C([H])=C(*)C([H])=C1F 0.000 claims description 2
- 125000001255 4-fluorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1F 0.000 claims description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- 239000005864 Sulphur Substances 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052711 selenium Inorganic materials 0.000 claims description 2
- 239000011669 selenium Substances 0.000 claims description 2
- 239000006285 cell suspension Substances 0.000 claims 1
- 238000002405 diagnostic procedure Methods 0.000 claims 1
- 210000004881 tumor cell Anatomy 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 15
- 238000011503 in vivo imaging Methods 0.000 abstract description 6
- 231100000419 toxicity Toxicity 0.000 abstract description 4
- 230000001988 toxicity Effects 0.000 abstract description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 3
- 239000000975 dye Substances 0.000 description 28
- 229940125904 compound 1 Drugs 0.000 description 26
- 229940125782 compound 2 Drugs 0.000 description 24
- 230000005284 excitation Effects 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 239000000523 sample Substances 0.000 description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 238000002372 labelling Methods 0.000 description 15
- 230000002438 mitochondrial effect Effects 0.000 description 12
- 238000010186 staining Methods 0.000 description 12
- BMZRVOVNUMQTIN-UHFFFAOYSA-N Carbonyl Cyanide para-Trifluoromethoxyphenylhydrazone Chemical compound FC(F)(F)OC1=CC=C(NN=C(C#N)C#N)C=C1 BMZRVOVNUMQTIN-UHFFFAOYSA-N 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 230000035515 penetration Effects 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000007850 fluorescent dye Substances 0.000 description 10
- 150000003839 salts Chemical class 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 230000004807 localization Effects 0.000 description 9
- 238000002311 multiphoton fluorescence microscopy Methods 0.000 description 9
- 229920000858 Cyclodextrin Polymers 0.000 description 8
- 241000289427 Didelphidae Species 0.000 description 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 8
- 238000000799 fluorescence microscopy Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 210000003292 kidney cell Anatomy 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 125000001188 haloalkyl group Chemical group 0.000 description 6
- 230000025608 mitochondrion localization Effects 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000012800 visualization Methods 0.000 description 6
- 125000006702 (C1-C18) alkyl group Chemical group 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 125000004491 isohexyl group Chemical group C(CCC(C)C)* 0.000 description 5
- 210000004731 jugular vein Anatomy 0.000 description 5
- 210000001700 mitochondrial membrane Anatomy 0.000 description 5
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 229940126214 compound 3 Drugs 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 125000005843 halogen group Chemical group 0.000 description 4
- 125000005647 linker group Chemical group 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 4
- 231100000057 systemic toxicity Toxicity 0.000 description 4
- XARJCDAOYQLEEQ-UHFFFAOYSA-M (2E)-2-[(2Z,4E)-5-(3,3-dimethyl-1-propylindol-1-ium-2-yl)-3-(4-nitrophenyl)penta-2,4-dienylidene]-3,3-dimethyl-1-propylindole iodide Chemical compound [I-].CC1(C(=[N+](C2=CC=CC=C12)CCC)/C=C/C(=C/C=C\1/N(C2=CC=CC=C2C/1(C)C)CCC)/C1=CC=C(C=C1)[N+](=O)[O-])C XARJCDAOYQLEEQ-UHFFFAOYSA-M 0.000 description 3
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 3
- 125000005917 3-methylpentyl group Chemical group 0.000 description 3
- 230000002407 ATP formation Effects 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 229910006069 SO3H Inorganic materials 0.000 description 3
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 125000003545 alkoxy group Chemical group 0.000 description 3
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 3
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 229910052792 caesium Inorganic materials 0.000 description 3
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 3
- 239000010941 cobalt Substances 0.000 description 3
- 229910017052 cobalt Inorganic materials 0.000 description 3
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 3
- 229940125898 compound 5 Drugs 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 229910052731 fluorine Inorganic materials 0.000 description 3
- 239000011737 fluorine Substances 0.000 description 3
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 3
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 3
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 3
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 3
- 229910052744 lithium Inorganic materials 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 3
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 239000002096 quantum dot Substances 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 150000003297 rubidium Chemical class 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 3
- 125000005913 (C3-C6) cycloalkyl group Chemical group 0.000 description 2
- 125000005918 1,2-dimethylbutyl group Chemical group 0.000 description 2
- XTWZIHLYYBWUKD-UHFFFAOYSA-N 2-(4-fluorophenyl)propanedial Chemical compound FC1=CC=C(C(C=O)C=O)C=C1 XTWZIHLYYBWUKD-UHFFFAOYSA-N 0.000 description 2
- AXZKOZLDEWIJPX-UHFFFAOYSA-N 2-(4-nitrophenyl)propanedial Chemical compound [O-][N+](=O)C1=CC=C(C(C=O)C=O)C=C1 AXZKOZLDEWIJPX-UHFFFAOYSA-N 0.000 description 2
- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 2
- 125000006176 2-ethylbutyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(C([H])([H])*)C([H])([H])C([H])([H])[H] 0.000 description 2
- 125000004493 2-methylbut-1-yl group Chemical group CC(C*)CC 0.000 description 2
- 125000005916 2-methylpentyl group Chemical group 0.000 description 2
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 2
- GJCOSYZMQJWQCA-UHFFFAOYSA-N 9H-xanthene Chemical compound C1=CC=C2CC3=CC=CC=C3OC2=C1 GJCOSYZMQJWQCA-UHFFFAOYSA-N 0.000 description 2
- 239000012099 Alexa Fluor family Substances 0.000 description 2
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 description 2
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- MDFFNEOEWAXZRQ-UHFFFAOYSA-N aminyl Chemical compound [NH2] MDFFNEOEWAXZRQ-UHFFFAOYSA-N 0.000 description 2
- UGUUDTWORXNLAK-UHFFFAOYSA-N azidoalcohol Chemical compound ON=[N+]=[N-] UGUUDTWORXNLAK-UHFFFAOYSA-N 0.000 description 2
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 2
- 229960004853 betadex Drugs 0.000 description 2
- 125000004181 carboxyalkyl group Chemical group 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 125000001309 chloro group Chemical group Cl* 0.000 description 2
- 238000011278 co-treatment Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- FHHZOYXKOICLGH-UHFFFAOYSA-N dichloromethane;ethanol Chemical compound CCO.ClCCl FHHZOYXKOICLGH-UHFFFAOYSA-N 0.000 description 2
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 2
- 125000004982 dihaloalkyl group Chemical group 0.000 description 2
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 239000003480 eluent Substances 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002284 excitation--emission spectrum Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000001506 fluorescence spectroscopy Methods 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- GDSRMADSINPKSL-HSEONFRVSA-N gamma-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO GDSRMADSINPKSL-HSEONFRVSA-N 0.000 description 2
- 229940080345 gamma-cyclodextrin Drugs 0.000 description 2
- 210000002288 golgi apparatus Anatomy 0.000 description 2
- 150000004820 halides Chemical class 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000002601 intratumoral effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 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 2
- 238000011068 loading method Methods 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 125000006682 monohaloalkyl group Chemical group 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 125000000018 nitroso group Chemical group N(=O)* 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 201000008968 osteosarcoma Diseases 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 125000003538 pentan-3-yl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])[H] 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 125000006684 polyhaloalkyl group Polymers 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000000512 proximal kidney tubule Anatomy 0.000 description 2
- 125000000714 pyrimidinyl group Chemical group 0.000 description 2
- 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 2
- 239000001022 rhodamine dye Substances 0.000 description 2
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 125000000335 thiazolyl group Chemical group 0.000 description 2
- 125000004001 thioalkyl group Chemical group 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 125000005389 trialkylsiloxy group Chemical group 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UIXKVNSFMFRIJK-UHFFFAOYSA-M (2Z)-2-[(2Z,4E)-3-(4-fluorophenyl)-5-(3-methyl-1,3-benzothiazol-3-ium-2-yl)penta-2,4-dienylidene]-3-methyl-1,3-benzothiazole iodide Chemical compound [I-].FC1=CC=C(C=C1)\C(=C/C=C/1\SC2=C(N\1C)C=CC=C2)\C=C\C=1SC2=C([N+]=1C)C=CC=C2 UIXKVNSFMFRIJK-UHFFFAOYSA-M 0.000 description 1
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 1
- PYKUGMFQWGRMHS-UHFFFAOYSA-N 1,2,3,3-tetramethyl-2h-indole;hydroiodide Chemical compound [I-].C1=CC=C2C(C)(C)C(C)[NH+](C)C2=C1 PYKUGMFQWGRMHS-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- PFVULLQPBZFZBR-UHFFFAOYSA-N 2,3,3-trimethyl-1-propyl-1,2-dihydroindol-1-ium iodide Chemical compound [I-].C1=CC=C2[NH+](CCC)C(C)C(C)(C)C2=C1 PFVULLQPBZFZBR-UHFFFAOYSA-N 0.000 description 1
- VZRIFNLQJXPRJI-UHFFFAOYSA-M 2,3-dimethyl-1,3-benzothiazol-3-ium;iodide Chemical compound [I-].C1=CC=C2[N+](C)=C(C)SC2=C1 VZRIFNLQJXPRJI-UHFFFAOYSA-M 0.000 description 1
- UGRAXKCTCJHEGH-UHFFFAOYSA-M 2-methyl-3-propyl-1,3-benzothiazol-3-ium;iodide Chemical compound [I-].C1=CC=C2[N+](CCC)=C(C)SC2=C1 UGRAXKCTCJHEGH-UHFFFAOYSA-M 0.000 description 1
- YHGCOZIRRUPMPX-UHFFFAOYSA-N 3,3-dimethyl-1-propylindol-1-ium Chemical compound C1=CC=C2[N+](CCC)=CC(C)(C)C2=C1 YHGCOZIRRUPMPX-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920001450 Alpha-Cyclodextrin Polymers 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 101150050192 PIGM gene Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 125000003342 alkenyl group Chemical group 0.000 description 1
- 229940043377 alpha-cyclodextrin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000002178 anthracenyl group Chemical group C1(=CC=CC2=CC3=CC=CC=C3C=C12)* 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000008135 aqueous vehicle Substances 0.000 description 1
- 125000004104 aryloxy group Chemical group 0.000 description 1
- 125000003785 benzimidazolyl group Chemical group N1=C(NC2=C1C=CC=C2)* 0.000 description 1
- 125000004604 benzisothiazolyl group Chemical group S1N=C(C2=C1C=CC=C2)* 0.000 description 1
- 125000004603 benzisoxazolyl group Chemical group O1N=C(C2=C1C=CC=C2)* 0.000 description 1
- 125000000499 benzofuranyl group Chemical group O1C(=CC2=C1C=CC=C2)* 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical compound C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 description 1
- 125000001164 benzothiazolyl group Chemical group S1C(=NC2=C1C=CC=C2)* 0.000 description 1
- 125000004196 benzothienyl group Chemical group S1C(=CC2=C1C=CC=C2)* 0.000 description 1
- 125000004541 benzoxazolyl group Chemical group O1C(=NC2=C1C=CC=C2)* 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 125000004218 chloromethyl group Chemical group [H]C([H])(Cl)* 0.000 description 1
- 125000000259 cinnolinyl group Chemical group N1=NC(=CC2=CC=CC=C12)* 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- XLJMAIOERFSOGZ-UHFFFAOYSA-M cyanate Chemical compound [O-]C#N XLJMAIOERFSOGZ-UHFFFAOYSA-M 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000004210 cyclohexylmethyl group Chemical group [H]C([H])(*)C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 125000006003 dichloroethyl group Chemical group 0.000 description 1
- 125000004774 dichlorofluoromethyl group Chemical group FC(Cl)(Cl)* 0.000 description 1
- 125000004772 dichloromethyl group Chemical group [H]C(Cl)(Cl)* 0.000 description 1
- 125000006001 difluoroethyl group Chemical group 0.000 description 1
- 125000001028 difluoromethyl group Chemical group [H]C(F)(F)* 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- GTSMOYLSFUBTMV-UHFFFAOYSA-N ethidium homodimer Chemical compound [H+].[H+].[Cl-].[Cl-].[Cl-].[Cl-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2C(C)=[N+]1CCCNCCNCCC[N+](C1=CC(N)=CC=C1C1=CC=C(N)C=C11)=C1C1=CC=CC=C1 GTSMOYLSFUBTMV-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 1
- 125000003709 fluoroalkyl group Chemical group 0.000 description 1
- 125000004785 fluoromethoxy group Chemical group [H]C([H])(F)O* 0.000 description 1
- 125000004216 fluoromethyl group Chemical group [H]C([H])(F)* 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 125000004438 haloalkoxy group Chemical group 0.000 description 1
- 125000006343 heptafluoro propyl group Chemical group 0.000 description 1
- 150000002390 heteroarenes Chemical class 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 125000003453 indazolyl group Chemical group N1N=C(C2=C1C=CC=C2)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 150000002513 isocyanates Chemical class 0.000 description 1
- 125000000904 isoindolyl group Chemical group C=1(NC=C2C=CC=CC12)* 0.000 description 1
- 125000001786 isothiazolyl group Chemical group 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 125000000842 isoxazolyl group Chemical group 0.000 description 1
- 238000010859 live-cell imaging Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 230000010280 mitochondria-mediated cell death Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000001715 oxadiazolyl group Chemical group 0.000 description 1
- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 125000006340 pentafluoro ethyl group Chemical group FC(F)(F)C(F)(F)* 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 208000007578 phototoxic dermatitis Diseases 0.000 description 1
- 231100000018 phototoxicity Toxicity 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000003226 pyrazolyl group Chemical group 0.000 description 1
- 125000005495 pyridazyl group Chemical group 0.000 description 1
- 125000001422 pyrrolinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 125000002294 quinazolinyl group Chemical group N1=C(N=CC2=CC=CC=C12)* 0.000 description 1
- 125000001567 quinoxalinyl group Chemical group N1=C(C=NC2=CC=CC=C12)* 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000001113 thiadiazolyl group Chemical group 0.000 description 1
- ACOJCCLIDPZYJC-UHFFFAOYSA-M thiazole orange Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1.C1=CC=C2C(C=C3N(C4=CC=CC=C4S3)C)=CC=[N+](C)C2=C1 ACOJCCLIDPZYJC-UHFFFAOYSA-M 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 125000004306 triazinyl group Chemical group 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- 125000003866 trichloromethyl group Chemical group ClC(Cl)(Cl)* 0.000 description 1
- 125000004385 trihaloalkyl group Chemical group 0.000 description 1
- 238000000482 two photon fluorescence microscopy Methods 0.000 description 1
- OIHZGFWAMWHYPA-UHFFFAOYSA-N xanthylium Chemical compound C1=CC=CC2=CC3=CC=CC=C3[O+]=C21 OIHZGFWAMWHYPA-UHFFFAOYSA-N 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
- C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/12—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being branched "branched" means that the substituent on the polymethine chain forms a new conjugated system, e.g. most trinuclear cyanine dyes
-
- 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
- G01N33/5005—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
- G01N33/5079—Mitochondria
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
An imaging method comprising bringing a biological specimen into association with a compound of Formula I; irradiating the specimen with light; and observing two-photon excited fluorescence images emitted from the compound of Formula I; Wherein X and Y is as defined herein; B is an optionally substituted aromatic or heteroaromatic ring; M is 1-3; p is 1-3; Z is an anion having a negative charge of n; n is 1-3; q is 1-3; provided that m x p = n x q. B may be selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl, particularly 4-pyridyl. m, p, n and q are preferably 1. Z may be a halide anion, particularly iodide. Compounds of Formula 1 wherein B is phenyl substituted with 1-3 groups independently selected from fluoro and C1-C3, particularly trifluoromethyl are disclosed. The disclosed compounds are indicated to have greater imaging depth and photostability than known agents, whilst having lower toxicity. The methods are disclosed to have selectivity for mitochondria. The methods are disclosed to have utility in both in vitro and in vivo imaging, in particular, imaging of tumours.
Description
The invention relates, in one aspect to methods of imaging biological samples in vitro and in vivo, to methods of imaging and diagnosis, and of compounds useful in such methods.
Introduction
Fluorescence imaging is used as a non-destructive way of tracking or analysing biological molecules, both in vitro and in vivo. Certain small molecules or proteins present in cells are intrinsically fluorescent (such as NADH and tryptophan). Alternatively, biological molecules can be labelled with an extrinsic fluorophore. Examples of extrinsic fluorophores include fluorescent antibodies, proteins and small molecule probes. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
Two-photon fluorescence microscopy is a technique that allows imaging of living tissue to a greater depth than conventional (single photon) fluorescence imaging. Being a type of multiphoton fluorescence, it employs red-shifted excitation light which is capable of exciting fluorescent dyes. Two-photon excitation is a phenomenon in which two photons are simultaneously absorbed by the same fluorophore by exposure to high photon density per unit volume and time by irradiation with a suitable light source. The use of infrared light minimizes scattering in the tissue. The background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced phototoxicity.
Probes for use in two-photon microscopy are known. These include coumarin, rhodamine, xanthene, and cyanine dyes. Examples of rhodamine dyes include Alexa Fluor® 488 (J Biol Chem (2004) 279:37544-37550). Examples of cyanine dyes include Alexa Fluor® 647, available from Thermo Fisher Scientific of Waltham, Massachusetts, USA.
Mitochondria, an organelle found in almost all eukaryotic cells, play a vital role in the life and death of cells. The function of mitochondria is to produce ATP. The production of ATP involves a series of electron transport systems in the oxidative phosphorylation pathway, which is also found to be associated with the generation of reactive oxygen species (ROS). The production of ROS in mitochondria leads to the propagation of free radicals, damaging cells, and contributing to cell death, which is known as mitochondria-mediated apoptosis.
Fluorescent probes which selectively stain mitochondria are powerful tools for monitoring and studying cellular processes. Such probes must be photostable under the conditions of light exposure from fluorescent microscopes. Fluorescent dyes for mitochondrial staining have been developed, including those mentioned above. The photostability of known dyes is, however, rather poor. Moreover, known probes display significant systemic toxicity, rendering them less useful for in vivo imaging.
In view of the foregoing, there is an urgent need to develop two-photon fluorescent probes that can selectively stain mitochondria under in vivo and in vitro conditions. The ideal characteristics of such an agent would include one or more of the following: i) narrow excitation and fluorescence emission spectra; ii) minimal fluorescence in an aqueous environment, iii) maximal fluorescence after incorporation in the lipophilic environment; and iv) high binding selectivity for live cells and tissue; v) high binding photostability, and vi) low systemic toxicity.
Fluorescent probes displaying selectivity for cancer cells are extremely desirable. Improved visualization of tumours in vivo would aid diagnosis, facilitate surgical resection, investigate therapeutic efficacy, and improve prognosis. Fluorescence imaging has high specificity and sensitivity and has been utilized for medical imaging. The present invention addresses these, and other, problems.
Brief Description of the Figures
Figure 1 shows mitochondrial localization of compound 1 in opossum kidney cells in vitro.
Figure 2 shows mitochondrial localization of compound 2 in opossum kidney cells in vitro.
Figure 3 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 1.
Figure 4 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 2.
Figure 5 shows an excitation scan of compound 1 at a penetration depth of 25 pm.
Figure 6 shows an excitation scan of compound 2 at 25 pm penetration depth.
Figure 7 shows podocyte labelling in glomeruli in a living mouse by compound 1.
Figure 8 shows accumulation of compounds of the invention in mitochrondria of U-2 OS (human osteosarcoma) cells, compared with prior art compounds.
Figure 9 shows localization after intratumoral (top) and intravenous application (bottom) of Compound 7 of the invention in nu/nu mice.
Summary of the Invention
According to a first aspect, the invention provides compounds of Formula I,
wherein X is selected from the group consisting of
Y is selected from the group consisting of
A and A’ are independently selected substituents;
B is an optionally substituted aromatic or heteroaromatic group;
R1-R12 are independently selected from hydrogen and a substituent;
Q and Q’ independently selected from NH, N(Ci-C6 alkyl), oxygen, sulphur, selenium and di(Ci-Ce alkyl)-methylene, * indicates the point of attachment of a group to the remainder of the molecule; m is an integer 1, 2 or 3;
p is an integer 1,2 or 3;
Z is an anion having a negative charge of n, n is an integer 1,2 or 3;
q is an integer 1,2 or 3; provided that mxp=n*q for use as a two-photon probe for fluorescence imaging.
According to a second aspect, the invention provides an imaging method comprising bringing a biological specimen into association with a compound of formula I, irradiating the specimen with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.
According to a third aspect, the invention provides a method for imaging live cells, the method comprising administering to an animal a compound of formula I, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.
According to a fourth aspect, the invention provides a class of compounds of formula I as defined wherein B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl, preferably trifluoromethyl.
Detailed Description and Preferred Embodiments
The invention relates to two-photon fluorescent dyes based of Formula I, which, in one embodiment, are employed as fluorescent probes for deep multiphoton imaging. The probes enable long-term real-time monitoring of in vivo samples and selective labelling of podocytes in glomerulus in living animals. The two-photon fluorescent dyes of the present invention can be employed for imaging of living samples to exceptionally large depth of up to 160 pm for periods of 90 minutes or longer.
Problems of the prior art are solved with polymethinium salts of Formula I, which show very strong fluorescence intensity, high target affinity, low systemic toxicity, high photostability and enable imaging to a significant penetration depth. The polymethinium salts of Formula I of the present invention can be used as two-photon probes for deep real-time imaging for podocytes in glomerulus, and cellular and subcellular visualization in general. These systems are based on the utilization of a structural motif of pentamethinium salts prepared from corresponding malondialdehydes.
The preparation of polymethinium salts of Formula I is based on condensation of a suitable aromatic malondialdehyde with a salt of the corresponding heteroaromatic compound. The syntheses has been described in Czech patent application 304094 (PV 2011-782); Chem. Commun. 2008,1901-1903; Bioconjug. Chem. 2013, 24,14451454; and Dyes Pigm. 2014, 107, 51-59, which are incorporated herein by reference.
Preferred compound for uses of the invention are those wherein X is a group wherein A, Q and Ri to R4 are as defined elsewhere herein.
Preferred compound for uses of the invention are those wherein Y is a group
wherein A’, Q’ and R7 to R10 are as defined elsewhere herein.
Preferably, B is a group selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl, optionally substituted as defined. More preferably, B is a pyridyl group. Still more preferably, B is a 4-pyridyl group. Most preferably, B is an unsubstituted 4-pyridyl group.
In a preferred class of compounds, B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl. In one embodiment, B is phenyl substituted with from one to three fluoro groups. In an alternative, preferred embodiment, B is phenyl substituted with from one to three (preferably one) trifluoromethyl groups. Specifically preferred values of B are 4-fluorophenyl, 3,5-difluorophenyl, 4-trifluoromethyl and 3-trifluoromethyl. Such compounds offer unusually high photostability and duration of imaging.
Preferably, A is selected from a C1-C18 alkyl group, or a group having the formula
(CH2)n1
wherein n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R13 is selected from the group consisting of C1-C18 alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, cesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an α, β or y cyclodextrin.
More preferably, A is selected from a C1-C18 alkyl group. More preferably, A is a C1Ce alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertbutyl, pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 4methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2 dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3dimethylbutyl, and 2-ethylbutyl. Most preferably, A is n-butyl.
Preferably, A’ is selected from a Ci-Cis alkyl group, or a group having the formula
(CH2)n1
wherein n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R™ is selected from the group consisting of Ci-Cis alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, caesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an a, β or γ cyclodextrin.
More preferably, A’ is selected from a C1-C18 alkyl group. More preferably, A is a CiCe alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertbutyl, pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 4methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3dimethylbutyl, and 2-ethylbutyl. Most preferably, A’ is n-butyl.
Preferably groups R1-R12 (where present) are independently selected from hydrogen, halide, Ci-Ce alkyl, C3-C6 cycloalkyl, Ci-Cs alkoxy, Ci-Ce thioalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce haloalkyl, Ci-Ce trialkylsilyloxy, phenyl, NH2, NH(Ci-C6 alkyl), N(Ci-C6 alkyl)2, N+(Ci-C6 alkyl)3, carboxylic acid, C1-C6 carboxyalkyl, hydroxy, azide, nitro, nitroso, nitrile, cyanate, isocyanate, thiocyanate, isothiocyanate, thiol, or a group having the formula
(CH2)n1
wherein n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R13 is selected from the group consisting of C1-C18 alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, cesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an α, β or γ cyclodextrin.
Preferably, R1-R12 (where present) are selected from fluorine and hydrogen. More preferably R1-R12 are hydrogen.
Preferably, Z is selected from the group consisting of chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate and p-toluenesulfonate. More preferably, Z is selected from the group consisting of chloride, bromide and iodide. Most preferably, Z is iodide.
In certain embodiments, compounds of the formula I are partially fluorinated, that is at least some of the hydrogen atoms are replaced with fluorine. In other embodiments, the compounds are perfluorinated, i.e. all of the hydrogen atoms are replaced with fluorine. These compounds display enhanced photostability.
A preferred class of compounds is those having the formula
Znwherein A, B, Q, Z and n are as defined elsewhere herein.
A very preferred compound for use in the invention is compound 1, (2Z)-3-propyl-2[(2Z,4E)-5-(3-propyl-1,3-benzothiazol-3-ium-2-yl)-3-(4-pyridyl)penta-2,4-dienylidene]-
1,3-benzothiazole, having the formula
Compound 1 wherein Z is defined above. Preferably, Z is chloride, bromide or iodide, most preferably iodide.
An alternative, very preferred compound for use in the invention is compound 2, 2[(1 E,3Z,5E)-5-(3,3-dimethyl-1-propyl-indolin-2-ylidene)-3-(4-pyridyl)penta-1,3-dienyl]-
3,3-dimethyl-1-propyl-indol-1-ium
wherein Z is defined above. Preferably, Z is chloride, bromide or iodide, most preferably iodide.
The compounds of the invention have utility in two-photon imaging methods. The methods of the invention are applicable to both in vitro and in vivo imaging.
In one aspect, the invention relates to a method for imaging living cells, the method comprising administering to an animal a compound of formula I, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.
Preferably, the irradiating light has a wavelength of from 600 to 1300 nm. In some embodiments the irradiating light has a wavelength of between 600 and 700 nm, more preferably between 640 and 680 nm, such as about 640 nm, about 655 nm, or about 680nm. In alternative embodiments, the irradiating light has a wavelength of from 680 to 1300 nm, more preferably from 1100 to 1200 nm, such as about 1180 nm.
Compounds of formula I are suitably administered to a live animal (including human) subject in an aqueous vehicle. Compounds of formula I are preferably present at a concentration of between 10 μΜ and 1 mM, more preferably between 100 μΜ and 500 μΜ, still more preferably 200 μΜ and 300 μΜ, such as about 250 μΜ. In other embodiments, compounds of the invention are employed at working concentrations of from 1 to 100 nM, preferably from 10 to 50 nM. This compares very favourably with mitochondrial probes known in the art, such as MitoTrackers® (available from Thermo Fisher Scientific of Waltham, Massachusetts, USA), typically used at concentrations of 100-200 nM, and rhodamine dyes, typically used at concentrations of 500-1000 nM.
Compounds of formula I are suitably administered to a live animal via intravenous injection. However, other modes of administration are contemplated.
The methods of the invention can be used to image any organ, organelle or cellular structure in living tissue. Preferably, the methods are used for imaging the kidney, and in particular the glomerulus. The compounds of the invention show high affinity for the glomerulus, and in particular the podocyte of the glomerulus.
The methods of the invention are capable of providing images of living tissue to a substantial depth compared with known methods. For example, the methods of the invention are capable of providing images at a depth of at least 50 pm, more preferably at least 100 pm, still more preferably at least 150 pm.
A problem with known mitochondrial probes is that they exhibit an undesirable degree of nonspecific accumulation, and are frequently redistributed within the endoplasmic reticulum. The probes of the invention, namely the compounds of formula I, exhibit an extremely high degree of selectivity for mitochondria.
A further issue with known classes of imaging agent (coumarin, rhodamine, xanthene and cyanine dyes) is their toxicity, which limits their utility in diagnostic applications. The compounds of formula demonstrate low cytotoxicity, and may display lower systemic toxicity.
Compounds of formula I are particularly suitable for forming conjugates with a variety of species. In particular, the compounds may be attached (covalently, e.g. via a linker or otherwise) to various substrates such as biomolecules (including antibodies, oligonucleotides, enzymes, proteins, saccharides, etc.), aptamers, nanoparticles, drug molecules and polymers.
Conjugation of the compounds of formula I to the substrate may suitably be achieved via a chemical linker. Various linking strategies are known, and the skilled person will be able to select the appropriate one. A preferred class of linkers is the Nhydroxysuccinimide or succinimidyl ester. Alternative linkers are described e.g. in W02004018493, which is incorporated herein by reference.
The compounds of formula I of the present invention under appropriate conditions, are selectively sequestered in mitochondria.
Definitions
As used herein, the term “substituent” refers to a group other than hydrogen. Preferred substituents include alkyl (such as Ci-Ce), alkenyl (such as C2-C6), alkoxy (such as C1C6), aryl (such as C6-C12), aryloxy (such as C6-C12), heteroaryl, fluoroalkyl (such as C1Ce), perfluoroalkyl (such as C1-C6) and perfluoroaryl (such as C6-C12), unless otherwise stated.
As used herein, the term “aromatic group” group that contains any carbon-based aromatic group preferably having from 6 to 14 ring carbon atoms. Preferred aromatic groups are phenyl, naphthyl, biphenyl, and anthracenyl, with phenyl most preferred.
As used herein, the term “heteroaromatic group refers to a 5- or 6-membered monocyclic aromatic group wherein 1, 2, 3,4 ring heteroatoms independently selected from O, S and N; or to a 8- to 11 -membered bicyclic aromatic group wherein 1 , 2, 3, 4 or 5 ring heteroatoms independently selected from O, S and N.
Examples of 5- or 6-membered monocyclic heteroaromatic groups include pyrrolinyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyrimidinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl and pyrimidinyl. Examples of 8- to 1 1 - membered bicyclic heteroaromatic groups include 6H-thieno[2,3-b]pyrrolyl, imidazo[2,1-b][1,3]thiazolyl, imidazo[5,1b][1,3]thiazolyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, benzoxazolyl e.g. benzoxazol-2-yl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzothienyl, benzofuranyl, naphthridinyl, quinolyl, quinoxalinyl, quinazolinyl, cinnolinyl and isoquinolyl.
Where the aromatic or heteroaromatic groups are described as substituted, substituents are from one to three groups independently selected from CrCe alkyl, C3C6 cycloalkyl, C1-C6 alkoxy, C1-C6 thioalkyl, C2-C6 alkenyl, C2-C6 alkynyl, CrCe haloalkyl, Ci-Ce trialkylsilyloxy, phenyl, NH2, NH(Ci-C6 alkyl), N(Ci-C6 alkyl)2, N+(Cr Ce alkyl)3, carboxylic acid, Ci-Ce carboxyalkyl, halide, hydroxy, azide, nitro, nitroso, nitrile and thiol.
As used herein, the term alkyl, unless otherwise specified, refers to a saturated straight or branched hydrocarbon chain of typically Ci to Ce, and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3- methylpentyl, 2,2-dimethylbutyl, and
2,3-dimethylbutyl, and the like.
The term “alkoxy,” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy, hexyloxy, isohexyloxy, cyclohexyloxy, 2,2-dimethylbutoxy, and 2,3-dimethylbutoxy, and the like.
As used herein, the term haloalkyl refers to an alkyl as defined herein, which is substituted by one or more halo groups as defined herein. The haloalkyl can be monohaloalkyl, dihaloalkyl, trihaloalkyl, or polyhaloalkyl including perhaloalkyl. A monohaloalkyl can have one iodo, bromo, chloro or fluoro within the alkyl group. Chloro and fluoro are preferred. Dihaloalkyl and polyhaloalkyl groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyl. Examples of haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. A perhaloalkyl refers to an alkyl having all hydrogen atoms replaced with halo atoms, e.g, trifluoromethyl.
The term “haloalkoxy,” as used herein, refers to a haloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Examples include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, pentafluoroethoxy, heptafluoropropoxy, difluorochloromethoxy, dichlorofluoromethoxy, difluoroethoxy, difluoropropoxy, dichloroethoxy and dichloropropoxy and the like.
Preparation of a Labeling Solution
Compounds of formula I generally have low solubility in water. Typically a stock solution is prepared dissolving the reagent in an organic solvent (suitably DMSO, dimethylformamide, acetonitrile, dioxane, tetrahydrofuran etc). The labeling solution is prepared by diluting the stock solution into aqueous buffer to the desired labeling concentration.
The amount of dye in the labeling solution is the minimum amount required to yield detectable staining in the sample, without significant background fluorescence or undesired staining of other organelles. The amount of reagent required for staining eukaryotic mitochondria depends on the sensitivity required for staining of intracellular vs. cell-free mitochondria, the number of cells present, and the permeability of the cell membrane to the reagent.
Typically cells incubated with 10-50 nM labeling solution will require about 2 to 5 minutes to acheive fluorescent staining, with a fully stable signal that is reached in about 10 minutes. Higher concentrations achieve more rapid staining. The exact concentration of stain to be used is dependent upon the experimental conditions and the desired results. The skilled person is able to determine these using routine trial and error.
Before or after exposure to the inventive methods, the cells may optionally be treated with solvents to fix, and optionally permeabilize, the membranes. Various fixatives and fixation conditions are suitable for achieving this, for example formaldehyde, methanol, and ethanol. Suitably, fixation is accomplished by incubating in a 4% solution of formaldehyde for 15 minutes.
Co-Iocation with additional agents
Methods according to the present invention may be combined with the use of an additional visualization reagent. One or more additional visualization reagents may be used in conjunction with the compounds of Formula I. The additional visualization reagent may be used to stain the entire cell, or a cellular substructure by selection of an appropriate reagent with the desired degree of selectivity, such as a labeled antibody, labeled oligonucleotide, an enzymic activity, or other indicator for a specific cellular component or substructure such as the cytoplasm, nucleus, membrane, lysosome, or Golgi apparatus. The imaging methods of the present invention and the response of the additional visualization reagent may be observed simultaneously or sequentially.
One class of appropriate additional detection reagents is fluorescent nucleic acid stains. A wide variety of appropriate nucleic acid stains are known, such as thiazole orange, propidium iodide, ethidium homodimer, and DAPI.
In another embodiment of the invention, an appropriate additional detection reagent is any probe that selectively stains a cellular organelle such as the cell membrane, nucleus, Golgi apparatus, endoplasmic reticulum, lysosomes, or a second mitochondrial stain, such as MitoTracker, Alexa-647 or Alexa-488.
Examples
Synthesis Examples
Example 1 - Synthesis of (2Z)-2-[(2Z,4E)-3-(4-fluorophenyl)-5-(3-methyl-1,3benzothiazol-3-ium-2-yl)penta-2,4-dienylidene]-3-methyl-1,3-benzothiazole iodide (Compound 3)
Compound 3
The flask was charged with 2-(4-fluorophenyl) malondialdehyde (166 mg, 1.0 mmol), 2,3-dimethyl-benzothiazolium iodide (611 mg, 2.1 mmol) and dry n-butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The product was separated by filtration and obtained solid was washed with ethanol (3x5 mL) and dried in vacuum. The crude product was recrystallized from ethanol-dichlormethane. Compound 3 was isolated as green solid. Yield of product was 323 mg, 55%.
Ή NMR (500 MHz, DMSO) δ: 8.02 (d, J= 7.9 Hz, 2H), 7.95 (d, J= 13.5 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.54 (t, J = 7.7 Hz, 2H), 5.82 (m, 6H), 3.61 (s, 6H); HRMS: calculated for C27H22FN2S2+ = 457.12029, found: 457.12075.
Example 2 - Synthesis of 2-[(1 E,3Z,5E)-3-(4-fluorophenyl)-5-(1,3,3-trimethylindolin2-ylidene)penta-1,3-dienyl]-1,3,3-trimethyl-indol-1-ium iodide (compound 4)
The flask was charged with 2-(4-fluorophenyl) malondialdehyde (166 mg, 1.0 mmol), 1-propyl-2,3,3-trimethylindol iodide (691 mg, 2.1 mmol) and dry n-butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The crude product was purified by column chromatography on silica (4x30 cm, eluent dichlormethane-methanol 10:1). Compound 4 was isolated as green solid. The yield of product was 345 mg, 57%.
Ή NMR (500 MHz, DMSO) δ: 8.45 (d, J = 14.2 Hz, 2H), 7.64 (d, J = 7.0 Hz, 2H), 7.36 (m, 8H), 7.26 (t, J= 7.2 Hz, 2H), 5.60 (m, J= 14.0 Hz, 2H), 3.33 (s, 6H), 1.74 (s, 12H); HRMS: calculated for C33H34FN2+ = 477.27005, found: 477.27026.
Example 3 - Synthesis of 2-((1 E,3Z,5Z)-3-(4-nitrophenyl)-5-(3-propyl-1,3benzothiazol-2-ylidene)penta-1,3-dienyl]-3-propyl-1,3-benzothiazol-3-ium iodide (compound 5)
no2
Compound 5
The flask was charged with 2-(4-nitrophenyl) malondialdehyde (193 mg, 1.0 mmol), 2-methyl-3-propylbenzothiazolium iodide (670 mg, 2.1 mmol) and dry n butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The product was separated by filtration and the obtained solid was washed with ethanol (3x5 mL) and dried in vacuum. The crude product was recrystalized from ethanol-dichlormethane. Compound 5 was isolated as a green solid. The yield of product was 377 mg, 62%.
Example 4 - Synthesis of (2E)-2-[(2Z,4E)-5-(3,3-dimethyl-1-propyl-indol-1-ium-2-yl)3-(4-nitrophenyl)penta-2,4-dienylidene]-3,3-dimethyl-1-propyl-indoline iodide (compound 6)
The flask was charged with 2-(4-nitrophenyl) malondialdehyde (193 mg, 1.0 mmol), 1,2,3,3-tetramethylindol iodide (632 mg, 2.1 mmol) and dry n-butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The product was purified by column chromatography on silica (4x30 cm, eluent dichlormethane-methanol 10:1). Compound 6 was isolated as a green solid. The yield of product was 387 mg, 61%.
Example 5
The following compounds were prepared in a similar manner to Examples 1 to 4.
Intracellular study
Gamma-substituted polymethinium salts 1 and 2 were tested for in vitro and in vivo 5 fluorescence microscopy applications in conventional wide-field, confocal and multiphoton fluorescence microscopy techniques. Both compounds showed selective localization in mitochondria of various cell lines, stated by the example of opossum kidney cells. In addition, these polymethinium salts are cell-permanent, with compound 1 being retained in mitochondria even after treatment with an uncoupling io reagent, e.g. carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which enables media exchange, fixation or even permeabilization of cells without loss of the staining (see Example 1). Compound 2 is not retained after FCCP staining, nevertheless it offers a broad application in long-term live-cell imaging. Both compounds did not show any signs of cytotoxicity in in vitro imaging experiments.
The newly prepared, positively charged symmetric polymethinium salts have convenient spectroscopic properties: narrow excitation and fluorescence emission spectra, minimal fluorescence in water environment, maximal fluorescence after incorporation in the lipophilic environment of mitochondrial membranes. Moreover, these dyes show extraordinary photostability, the significantly outperform commercially available xanthylium dyes broadly used for mitochondrial labelling.
Additionally and more importantly, both dyes can serve for in vivo imaging. Compounds 1 and 2 are two-photon fluorescent probes, which were successfully employed for podocyte labelling in glomeruli in living mice of C57BI/6J-Rj (see Examples 7-11). Compound 1 enables in vivo imaging for 90 minutes or longer, compound 2 up to several hours and was very well tolerated for several hours, and also with reinjection. In terms of the depth penetration, compound 1 outperforms commercially available Alexa-647-albumin for in vivo imaging, compound 1 enables imaging up to 160 pm (in contrast to Alexa-647 conjugate to 120 pm)
Animals injected with compound 1 died after about 1 to 1 1Λ hours. This indicates toxicity but no proof was found for direct toxicity in the kidney (n=2).
Compound 2 also demonstrated excitation at around 800 nm
Compound 1 is suitable for deep tissue imaging and was superior to Alexa 647Albumin.
Compound 2 was able to demonstrate long term imaging for several hours.
In vitro mitochondrial localization of compound 1: Independence on the mitochondrial membrane potential
Opossum kidney cell (OK) line was used for in vitro evaluation of the localization and fluorescence microscopy performance of compound 1. Compound 1 was incubated at 50 nM concentration with the cells for 30 minutes at physiological conditions and washed with phosphate buffered saline, then the cells were imaged using a Leica SP8 confocal fluorescence microscope equipped with a white light laser. The excitation wavelength employed for compound 1 was 655 nm. HyD-Detectors were used for image acquisition. Uncoupling was performed by 30 second treatment with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM - 10 μΜ) disrupting ATP synthesis.
Figure 1 shows mitochondrial localization of compound 1 in Opossum kidney cells in vitro, specifically:
Figure 1A) Mitochondria labelled with 50 nM compound 1 (before uncoupler treatment)
Figure 1B) co-treatment with a mitochondrial uncoupler FCCP
Figure 1C) tetramethylrhodamine methyl ester (TMRM) localization in opossum cells, Figure 1D) TMRM co-treated with FCCP uncoupler.
Compound 1 did not show any obvious signs of cytotoxicity.
Based on the localization of the dyes, it is obvious that, in contrast to a commercial mitochondria-specific dye TMRM (significantly decreased fluorescence intensity), localization of compound 1 based on a gamma-substituted polymethinium salt is quite independent of the mitochondrial membrane potential, since the mitochondrial staining is retained even after the FCCP treatment.
It is possible to combine mitochondrial labelling with compound 1 with staining using, for example, fluorescent proteins, labeled antibodies, nanobodies, quantum dots, other small organelles probes.
Example 6
In vitro mitochondrial localization of compound 2: mitochondrial membrane potential dependence
Opossum kidney cell (OK) line was used for in vitro evaluation of the localization and fluorescence microscopy performance of compound 2. Compound 2 was incubated at 50 nM concentration with the cells for 30 minutes at physiological conditions and washed with phosphate buffered saline, then the cells were imaged by Leica SP8 confocal fluorescence microscope equipped with a white light laser. The excitation wavelength used for compound 2 was 640 nm. HyD-Detectors were used for image acquisition. Uncoupling was performed by a 30 second treatment with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM - 10 μΜ) disrupting ATP synthesis.
Figure 2 shows mitochondrial localization of compound 2 in opossum kidney cells in vitro, specifically:
Figure 2 A) Mitochondria labelled with 50 nM compound 2 (before uncoupler treatmnent)
Figure 2 B) 30 second co-treatment with a mitochondrial uncoupler FCCP, according to the decreased fluorescence signal (the same manner as for commercial TMRM mitochondrial dye).
Compound 2 did not show any obvious signs of cytotoxicity. Retention of compound 2 in mitochondria depends on the mitochondrial membrane potential.
It is possible to combine mitochondrial labelling with compound 2 with staining using, for example, fluorescent proteins, labeled antibodies, nanobodies, quantum dots, other small organelles probes.
Example 7
Compound 1 imaging from basolateral site: Multiphoton fluorescence microscopy in C57BI/6J-Ri mouse
Model mouse strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 1 was used at 250 μΜ concentration in 0.9% NaCI (volume 120 pl). The imaging was performed immediately after dye application using a custom-built multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength used was 1180 nm and the frame rate was 1 fps.
Figure 3 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 1 (excitation at 1180 nm, 1fps), specifically:
Figure 3A) Tissue image prior to dye loading,
Figure 3B) Tissue image 20 seconds after dye application, and
Figure 3C) Tissue image 6 minutes after dye application.
The target structure was stained 6 minutes after application of compound 1.
Example 8
Compound 2 imaging from basolateral site: Multiphoton fluorescence microscopy in C57BI/6J-Rj mouse
Model mouse strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 2 was used at
250 μΜ concentration in 0.9% NaCI (volume 120 μΙ). The imaging was performed immediately after dye application using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength used was 1180 nm and the frame rate was 1 fps.
Figure 4 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 2 (excitation at 1180 nm, 1fps), specifically:
Figure 4A) Tissue image prior to dye loading,
Figure 4B) Tissue image 90 seconds after dye application, and
Figure 4C) Tissue image 23 minutes after dye application.
Example 9
Excitation wavelength scan: Multiphoton fluorescence microscopy in C57BI/6J-Ri mouse by Compound 1
Model mouse strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 2 was used at 250 μΜ concentration in 0.9% NaCI (volume 120 μΙ). The imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength scan was performed at 25 pm depth (px size 228 nm).
Figure 5 shows an excitation scan of compound 1 at a penetration depth of 25 pm. Selected wavelengths (maximal fluorescence intensities): 820 (max 4000), 1100 (max 2500), 1150 (max 5000), 1200 (max 4000), 1300 (max 5000). Podocyte labelling in the glomerulus in a living mouse was achieved by the methods of the invention. (Key: PT = proximal tubule, CD = collecting duct).
Example 10
Excitation wavelength scan: Multiphoton fluorescence microscopy in C57BI/6J-Ri mouse by Compound 2
Mouse model strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 2 was used at 250 μΜ concentration in 0.9% NaCI (volume 120 μΙ). The imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength scan was performed at 25 pm depth (px size 381 nm).
Figure 6 shows an excitation scan of compound 2 at 25 pm penetration depth. Selected wavelengths (maximal fluorescence intensities): 820 (max 4000), 1100 (max 2500), 1150 (max 5000), 1200 (max 4000), 1300 (max 5000). Podocyte labelling in the glomerulus in a living mouse was achieved by the methods of the invention. (Key: PT = proximal tubule, CD = collecting duct).
Example 11
Multiphoton fluorescence in vivo microscopy in C57BI/6J-Ri mouse by Compound 1 Mouse model strain C57BI/6J-Rj was used for this experiment. The mice were catheterized using a jugular-vein catheter for dye application. Compound 1 was used at 250 pM concentration in 0.9% NaCI (volume 120 pl). The imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used.
Figure 7 shows podocyte labelling in glomeruli in a living mouse by polymethinium salt 1 at A) 133 pm and B) 160 pm penetration depth. The penetration depth exceeds the best-performing Alexa-647-Albumin, in which case only 120 pm penetration depth can be achieved.
It is possible to combine mitochondrial labelling with compound 1 with staining using, for example, fluorescent proteins, labeled antibodies, nanobodies, quantum dots, other small organelles probes.
Example 12 - UV-visible and fluorescence experiments
For UV-visible analysis, the absorption spectra in 6.25 pM of all studied compounds in DMSO were recorded over wavelengths of 300 - 800 nm using a GBC Cintra 404 spectrometer. The absorbance of these solutions was determined in a conventional 1 cm PMMA cell. The results are shown in Table 1.
Fluorescence spectroscopy studies were carried out using a SCINCO FluoroMate FS2 spectrometer. The compounds were dissolved in DMSO and the solution (39 nM) was placed in a 1 cm PMMA cell. The results are shown in Table. 1.
Amax (nm) | Aemmax (nm) | Aexmax (nm) | |
Compound 3 | 645 | 668.6 | 643.7 |
Compound 7 | 642 | 657.1 | 641.7 |
Compound 8 | 652 | 674.7 | 650.9 |
Compound 9 | 643 | 658.3 | 642.6 |
TABLE 1
Amax - maximum absorbance wavelength Aemmax - maximum emission wavelength Aexmax - maximum excitation wavelength
Example 13 - Co-localization experiment
A mitochondria-specific probe, MitoTracker Green, was used to confirm the selective accumulation in mitochrondria of U-2 OS (human osteosarcoma) cells. Cells were incubated with the compounds for 30 minutes in the presence of 100 nM MitoTracker Green, washed with phosphate buffer saline, and then imaged. The images were acquired using an inverse fluorescent microscope (Olympus IX-81).
Results are shown in Figure 8.
Example 14 - Effect of Compound 7 on localization in vivo
Nu/nu mice bearing subcutaneously growing human pancreatic carcinoma (MIA PaCa2) received Compound 7 intravenously. Compound 7 was also injected intratumorally in mice bearing human glioblastoma (U-87). Mice were anaesthetised and subjected to fluorescence imaging. Imaging was performed immediately after dye application, and then after 1 and 72 hours using Bruker In-Vivo Xtreme II.
Figure 9 shows localization after intratumoral (top) and intravenous application (bottom) of Compound 7 in nu/nu mice.
Claims (23)
1. An imaging method comprising:
bringing a biological specimen into association with a compound of formula I; irradiating the specimen with light; and observing two-photon excited fluorescence images emitted from the compound of formula I m+ [Zn']q
P
Formula I wherein X is selected from the group consisting of
A and A’ are independently selected substituents;
B is an optionally substituted aromatic or heteroaromatic ring
R1-R12 are independently selected from hydrogen and a substituent;
Q and Q’ independently selected from NH, N(Ci-Ce alkyl), oxygen, sulphur, selenium and di(Ci-C6)-methylene, * indicates the point of attachment of a group to the remainder of the molecule;
m is an integer 1, 2 or 3;
p is an integer 1,2 or 3;
Z is an anion having a negative charge of n, n is an integer 1,2 or 3;
q is an integer 1,2 or 3;
provided that mxp=nxq.
2. A method according to claim 1 wherein X is a group
Ft wherein A, Q and Ri to R4 are as defined in claim 1.
3. A method according to claim 1 or 2 wherein Y is a group wherein A’, Q’ and R7 to R10 are as defined in claim 1.
4. A method according to any preceding claim, wherein B is a group selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl.
5. A method according to claim 4 wherein B is a pyridyl group.
6. A method according to claim 5 wherein B is a 4-pyridyl group.
7. A method according to any preceding claim wherein A is selected from a Cr Cis alkyl group.
8. A method according to any preceding claim wherein A’ is selected from a Cr Cis alkyl group.
9. A method according to any preceding claim wherein R1-R12 (where present) are H.
10. A method according to any preceding claim wherein m is 1, p is 1, n is 1 and q is 1.
11. A method according to any preceding claim wherein Z is a halide anion, preferably iodide.
12. A compound of formula I as defined in any one of claims 1 to 3 or 7 to 11 wherein B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl, preferably trifluoromethyl.
13. A compound according to claim 12 wherein B is selected from 4-fluorophenyl, 3,5-difluorophenyl, 4-trifluoromethyl and 3-trifluoromethyl.
14. A compound according to claim 12 which is one of:
J ?
and
15. A compound of formula I as defined in any one of claims 1 to 14 for use in diagnostic methods practised on the human or animal body.
16. A method of imaging live cells, the method comprising administering to an animal a compound as defined in any one of claims 1 to 15, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.
17. The method of any one of claims 1 to 11 or 16 wherein the irradiating light has a wavelength of between 600 to 1300 nm.
18. The method of any one of claims 1 to 11, 16 or 17, wherein the cells to be imaged are at a depth of at least 100 pm.
19. The method of claim 18, wherein the cells to be imaged are at a depth of at least 150 pm.
20. The method of any one of claims 16 to 19 wherein the cells are podocytes.
21. The method according to any one of claims 16 to 20 wherein the imaging method is for detecting tumour cells.
22. A method for visualizing cellular organelles in tissue or in cell suspension, said method comprising the steps of:
(a) obtaining a tissue sample or cell sample, said sample containing a plurality of cells;
(b) contacting said tissue sample or cell sample with a compound of Formula I as defined in any of claims 1 to 14;
(c) irradiating the specimen with light; and (d) observing two-photon excited fluorescence images.
23. The method according to claim 22 wherein the organelle is a mitochondrion.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1707342.0A GB2567124A (en) | 2017-05-08 | 2017-05-08 | Imaging agents and methods |
PCT/EP2017/062048 WO2018206126A1 (en) | 2017-05-08 | 2017-05-18 | Imaging agents and methods |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1707342.0A GB2567124A (en) | 2017-05-08 | 2017-05-08 | Imaging agents and methods |
Publications (2)
Publication Number | Publication Date |
---|---|
GB201707342D0 GB201707342D0 (en) | 2017-06-21 |
GB2567124A true GB2567124A (en) | 2019-04-10 |
Family
ID=59065636
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB1707342.0A Withdrawn GB2567124A (en) | 2017-05-08 | 2017-05-08 | Imaging agents and methods |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB2567124A (en) |
WO (1) | WO2018206126A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023160737A1 (en) | 2022-02-23 | 2023-08-31 | Univerzita Karlova | Polymethinium salts as inhibitors of dihydroorotate dehydrogenase |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111574499B (en) * | 2020-06-09 | 2021-12-10 | 河南大学 | Cy5-650 and application thereof in preparation of antitumor drugs |
CN111675921B (en) * | 2020-06-09 | 2021-12-14 | 河南大学 | CYPY and application thereof in preparation of antitumor drugs |
CN113429335B (en) * | 2021-06-25 | 2023-05-16 | 安徽大学 | Lysosome-targeted dual-response two-photon fluorescent probe and preparation method and application thereof |
CN113773666A (en) * | 2021-08-11 | 2021-12-10 | 大连理工大学 | Triplet state intersystem crossing cyanine dye, preparation method and application thereof |
CN114045045B (en) * | 2021-10-26 | 2022-11-04 | 大连理工大学 | Single-photon up-conversion pentamethine cyanine photosensitive dye, and preparation method and application thereof |
CN114736200B (en) * | 2022-03-22 | 2024-08-16 | 大连理工大学 | Heavy atom-free triplet photosensitizer based on thiopentamethine cyanine dye, and preparation method and application thereof |
CN114874638B (en) * | 2022-06-23 | 2024-01-19 | 西安建筑科技大学 | Meso-position substituted pentamethine cyanine dye, preparation method and application thereof, and fluorescent probe |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007028118A2 (en) * | 2005-09-02 | 2007-03-08 | Visen Medical, Inc. | Nicotinic acid and picolinic acid derived near-infrared fluorophores |
WO2008109832A2 (en) * | 2007-03-08 | 2008-09-12 | Visen Medical, Inc. | Viable near-infrared fluorochrome labeled cells and methods of making and using same |
WO2010121163A2 (en) * | 2009-04-17 | 2010-10-21 | Li-Cor, Inc. | Fluorescent imaging with substituted cyanine dyes |
US20100268070A1 (en) * | 2008-11-26 | 2010-10-21 | Visen Medical, Inc. | Methods and Compositions for Identifying Subjects at Risk of Developing Stent Thrombosis |
WO2012054784A1 (en) * | 2010-10-20 | 2012-04-26 | Li-Cor, Inc. | Fluorescent imaging with substituted cyanine dyes |
WO2013148319A1 (en) * | 2012-03-30 | 2013-10-03 | Visen Medical, Inc. | Bacterial imaging agents and methods of using same |
WO2015066290A1 (en) * | 2013-10-31 | 2015-05-07 | Beth Israel Deaconess Medical Center | Near-infrared fluorescent contrast bioimaging agents and methods of use thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PT3147292T (en) | 2002-08-23 | 2018-11-22 | Illumina Cambridge Ltd | Labelled nucleotides |
CZ304094B6 (en) | 2011-12-01 | 2013-10-16 | Vysoká skola chemicko-technologická v Praze | Use of polymethine salts as mitochondrial probes |
-
2017
- 2017-05-08 GB GB1707342.0A patent/GB2567124A/en not_active Withdrawn
- 2017-05-18 WO PCT/EP2017/062048 patent/WO2018206126A1/en active Application Filing
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007028118A2 (en) * | 2005-09-02 | 2007-03-08 | Visen Medical, Inc. | Nicotinic acid and picolinic acid derived near-infrared fluorophores |
WO2008109832A2 (en) * | 2007-03-08 | 2008-09-12 | Visen Medical, Inc. | Viable near-infrared fluorochrome labeled cells and methods of making and using same |
US20100268070A1 (en) * | 2008-11-26 | 2010-10-21 | Visen Medical, Inc. | Methods and Compositions for Identifying Subjects at Risk of Developing Stent Thrombosis |
WO2010121163A2 (en) * | 2009-04-17 | 2010-10-21 | Li-Cor, Inc. | Fluorescent imaging with substituted cyanine dyes |
WO2012054784A1 (en) * | 2010-10-20 | 2012-04-26 | Li-Cor, Inc. | Fluorescent imaging with substituted cyanine dyes |
WO2013148319A1 (en) * | 2012-03-30 | 2013-10-03 | Visen Medical, Inc. | Bacterial imaging agents and methods of using same |
WO2015066290A1 (en) * | 2013-10-31 | 2015-05-07 | Beth Israel Deaconess Medical Center | Near-infrared fluorescent contrast bioimaging agents and methods of use thereof |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023160737A1 (en) | 2022-02-23 | 2023-08-31 | Univerzita Karlova | Polymethinium salts as inhibitors of dihydroorotate dehydrogenase |
Also Published As
Publication number | Publication date |
---|---|
GB201707342D0 (en) | 2017-06-21 |
WO2018206126A1 (en) | 2018-11-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
GB2567124A (en) | Imaging agents and methods | |
Niu et al. | Highly photostable two-photon NIR AIEgens with tunable organelle specificity and deep tissue penetration | |
Ren et al. | A general method to increase stokes shift by introducing alternating vibronic structures | |
US9315465B2 (en) | Photostable AIE luminogens for specific mitochondrial imaging and its method of manufacturing thereof | |
CN103003282A (en) | A process for the preparation of novel porphyrin derivatives and their use as PDT agents and fluorescence probes | |
Ma et al. | Design and synthesis of functionalized rhodamine based probes for specific intracellular fluorescence imaging of Fe3+ | |
Xu et al. | Targeting tumor hypoxia with 2-nitroimidazole-indocyanine green dye conjugates | |
Dong et al. | A reversible frequency upconversion probe for real-time intracellular lysosome-pH detection and subcellular imaging | |
US9182350B2 (en) | Naphthalene-based two-photon fluorescent probes, preparation method and use thereof | |
Zhang et al. | Synthesis, spectral properties of cell-permeant dimethine cyanine dyes and their application as fluorescent probes in living cell imaging and flow cytometry | |
Tian et al. | NAD (P) H-triggered probe for dual-modal imaging during energy metabolism and novel strategy of enhanced photothermal therapy in tumor | |
CN112500386B (en) | Near-infrared HClO fluorescent probe based on piroctone olamine, preparation and application thereof | |
Terrones et al. | A silica supported tricarbocyanine based pH nanosensor with a large Stokes shift and a near infrared fluorescence response: performance in vitro and in live cells | |
Chen et al. | Two novel two-photon excited fluorescent pH probes based on the A-π-D-π-A system for intracellular pH mapping | |
US8968997B2 (en) | Benzoxazole-based fluorescent metal ion indicators | |
ES2718835T3 (en) | Near infrared fluorescent dyes (NIR) | |
Fu et al. | A Raman/fluorescence dual-modal imaging guided synergistic photothermal and photodynamic therapy nanoplatform for precision cancer theranostics | |
Tang et al. | A new metal-free near-infrared fluorescent probe based on nitrofuran for the detection and bioimaging of carbon monoxide releasing molecule-2 in vivo | |
WO2018064961A1 (en) | Multi-modal bioprobe for bladder cancer imaging and photodynamic therapy | |
Patel et al. | Impact of Substituents in Tumor Uptake and Fluorescence Imaging Ability of Near‐Infrared Cyanine‐like Dyes | |
Bokan et al. | Switchable phenolo-cyanine reporters containing reactive alkylcarboxylic groups for fluorescence-based targeted drug delivery monitoring | |
KR101842633B1 (en) | Diagnostic or therapeutic composition of cancer which is activated by cathepsin B, and near-infrared imaging and phototherapy of tumor using the same | |
CN112694469B (en) | Pyrrosinyl-based HOCl fluorescent probe, and preparation method and application thereof | |
CN109180568A (en) | A kind of identification mitochondria and fluorescence probe and application with two-phpton property | |
Lin et al. | Revealing the dynamics of a mitochondrial microenvironment during apoptosis under two-photon fluorescence lifetime microscopy using a cyclic iridium (iii) complex |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |