Fluorescent Copper(II) Bis(thiosemicarbazonates): Synthesis, structures, electron paramagnetic resonance, radiolabeling, in vitro cytotoxicity and confocal fluorescence microscopy studies

S I Pascu, P A Waghorn, B W C Kennedy, R L Arrowsmith, S R Bayly, J R Dilworth, M Christlieb, R M Tyrrell, J Zhong, R M Kowalczyk, D Collison, P K Aley, G C Churchill, F I Aigbirhio

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Abstract

Copper bis(4-ethyl-3-thiosemicarbazonato) acenaphthenequinone (1) and copper bis(4-methyl-3-thiosemicarbazonato) acenaphthenequinone (2) are synthesized and characterized in solution, in the solid state, and radiolabeled. Serum-protein binding radio-assays show good stability in solution and about 25% binding to protein over 1 h, which is comparable with the hypoxia selective tracer [Cu-64(ATSM)]. Cyclic voltammetry shows fast and reversible reduction at redox potentials similar to the values known for hypoxia-selective copper compounds. However, despite this, complex 1. does not show any hypoxic-selective uptake in HeLa cells over 1-h standard assays. Possible reasons for this are studied by using the intrinsic fluorescence of the Cu-II complexes to determine the cellular distributions and uptake mechanism by confocal microscopy. The complexes are found to bind to the external cell membrane and disperse evenly in the cytoplasm only after a very slow cell internalization (> 1 h). No significant changes in distribution are observed by fluorescence imaging under hypoxic conditions. The rate of localization in the cytoplasm contrasts with their Zn-II analogues, which are known to have fast cell uptake (up to 20 min) and a clear localization in lysosomes and mitochondria. The cytotoxicity mechanism of 1 over 24 h against a number of adherent cell lines is seen to be by membrane disruption and is of a comparable magnitude to that of [Cu(ATSM)], as demonstrated by methyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays.
Original languageEnglish
Pages (from-to)506-519
Number of pages14
JournalChemistry - An Asian Journal
Volume5
Issue number3
DOIs
Publication statusPublished - Feb 2010

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Confocal microscopy
Fluorescence microscopy
Cytotoxicity
Paramagnetic resonance
Copper
Assays
Fluorescence
Copper compounds
Mitochondria
Cell membranes
L-Lactate Dehydrogenase
Cyclic voltammetry
Blood Proteins
Cells
Membranes
Imaging techniques
Proteins
acenaphthenequinone

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Fluorescent Copper(II) Bis(thiosemicarbazonates): Synthesis, structures, electron paramagnetic resonance, radiolabeling, in vitro cytotoxicity and confocal fluorescence microscopy studies. / Pascu, S I; Waghorn, P A; Kennedy, B W C; Arrowsmith, R L; Bayly, S R; Dilworth, J R; Christlieb, M; Tyrrell, R M; Zhong, J; Kowalczyk, R M; Collison, D; Aley, P K; Churchill, G C; Aigbirhio, F I.

In: Chemistry - An Asian Journal, Vol. 5, No. 3, 02.2010, p. 506-519.

Research output: Contribution to journalArticle

Pascu, SI, Waghorn, PA, Kennedy, BWC, Arrowsmith, RL, Bayly, SR, Dilworth, JR, Christlieb, M, Tyrrell, RM, Zhong, J, Kowalczyk, RM, Collison, D, Aley, PK, Churchill, GC & Aigbirhio, FI 2010, 'Fluorescent Copper(II) Bis(thiosemicarbazonates): Synthesis, structures, electron paramagnetic resonance, radiolabeling, in vitro cytotoxicity and confocal fluorescence microscopy studies', Chemistry - An Asian Journal, vol. 5, no. 3, pp. 506-519. https://doi.org/10.1002/asia.200900446
Pascu, S I ; Waghorn, P A ; Kennedy, B W C ; Arrowsmith, R L ; Bayly, S R ; Dilworth, J R ; Christlieb, M ; Tyrrell, R M ; Zhong, J ; Kowalczyk, R M ; Collison, D ; Aley, P K ; Churchill, G C ; Aigbirhio, F I. / Fluorescent Copper(II) Bis(thiosemicarbazonates): Synthesis, structures, electron paramagnetic resonance, radiolabeling, in vitro cytotoxicity and confocal fluorescence microscopy studies. In: Chemistry - An Asian Journal. 2010 ; Vol. 5, No. 3. pp. 506-519.
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abstract = "Copper bis(4-ethyl-3-thiosemicarbazonato) acenaphthenequinone (1) and copper bis(4-methyl-3-thiosemicarbazonato) acenaphthenequinone (2) are synthesized and characterized in solution, in the solid state, and radiolabeled. Serum-protein binding radio-assays show good stability in solution and about 25{\%} binding to protein over 1 h, which is comparable with the hypoxia selective tracer [Cu-64(ATSM)]. Cyclic voltammetry shows fast and reversible reduction at redox potentials similar to the values known for hypoxia-selective copper compounds. However, despite this, complex 1. does not show any hypoxic-selective uptake in HeLa cells over 1-h standard assays. Possible reasons for this are studied by using the intrinsic fluorescence of the Cu-II complexes to determine the cellular distributions and uptake mechanism by confocal microscopy. The complexes are found to bind to the external cell membrane and disperse evenly in the cytoplasm only after a very slow cell internalization (> 1 h). No significant changes in distribution are observed by fluorescence imaging under hypoxic conditions. The rate of localization in the cytoplasm contrasts with their Zn-II analogues, which are known to have fast cell uptake (up to 20 min) and a clear localization in lysosomes and mitochondria. The cytotoxicity mechanism of 1 over 24 h against a number of adherent cell lines is seen to be by membrane disruption and is of a comparable magnitude to that of [Cu(ATSM)], as demonstrated by methyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays.",
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AU - Kennedy, B W C

AU - Arrowsmith, R L

AU - Bayly, S R

AU - Dilworth, J R

AU - Christlieb, M

AU - Tyrrell, R M

AU - Zhong, J

AU - Kowalczyk, R M

AU - Collison, D

AU - Aley, P K

AU - Churchill, G C

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