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Two-Photon Microscopy of E-Combretastatin uptake and activation in live mammalian cells

Scherer, KM


KM Scherer


Cancer is one of the most common causes of death in the Western world. Hence, the
development of novel effective treatment modalities to combat this disease is important.
Combretastatins are powerful anticancer drugs that are being evaluated in phase I/II clinical
trials. Combretastatins act by binding to p-tubulin, preventing microtubule assembly and
inhibiting angiogenesis in developing tumours. Most widely studied is the natural product
c/5(Z)-combretastatin A4 that was originally isolated from the African bush willow
Combretum caffrum by Pettit and co-workers. Chemically Combretastatins are substituted
stilbenes, exhibiting major activity in the cis-form, whereas the corresponding fr-am'-isomers
are ~ 2 - 3 orders of magnitude less cytotoxic.
Combretastatins undergo reversible photoisomerization upon irradiation with UV/visible
light. Here a novel form of two-photon phototherapy is proposed, in which the "inactive"
fnms(£)-combretastatins are used as anticancer pro-drugs that may be activated (converted to
the cw-isomer) by localized irradiation with light at the tumour site. The use of near-infrared
(NIR) light to trigger a nonlinear two-photon excited photoactivation process provides deeper
light penetration into tissues and minimizes cellular autofluorescence compared with
UV/visible light.
The work described in this thesis is highly multidisciplinary. A major task was the synthesis
of a range of combretastatin derivatives as target compounds for the novel form of
two-photon excited anticancer phototherapy proposed herein. The cytotoxicity of the drug
candidates was assessed on live mammalian cell lines. Photochemical properties such as
fluorescence quantum yields, fluorescence lifetimes, photoisomerization quantum yields and
two-photon absorption characteristics of the combretastatin derivatives were investigated
spectroscopically. The fact that the trans-isomers fluoresce in most cases, whereas ds-isomers
are virtually non-fluorescent has allowed multiphoton fluorescence lifetime imaging (FLIM)
of the real-time uptake of the inactive pro-drugs into live mammalian cells, as well as
monitoring their intracellular distribution and concentration. Two-photon excitation of
^-Combretastatins on monolayers of live cells led to effective conversion of the inactive
pro-drugs to the highly cytotoxic Z-derivatives leading to apoptosis in the irradiated areas
within 24 h. In conclusion, the two-photon excited activation of £-combretastatins on live
cells was demonstrated, making £-combretastatins promising pro-drug candidates for
two-photon excited anticancer phototherapy.


Scherer, K. Two-Photon Microscopy of E-Combretastatin uptake and activation in live mammalian cells. (Thesis). Salford: University of Salford

Thesis Type Thesis
Deposit Date Mar 9, 2016
Publicly Available Date Sep 23, 2023

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