Fluorescence lifetime imaging of E-combretastatin uptake and distribution in live mammalian cells

Abstract

To investigate within live mammalian cells the uptake and disposition of combretastatins, fluorescence lifetime imaging was used with two-photon excitation (2PE). Combretastatin A4 (CA4) and analogues are potential anticancer drugs due to their ability to inhibit angiogenesis. E(trans)-combretastatins are considerably less active than the Z(cis)-combretastatins proposed for clinical use. However the E-combretastatins exhibit stronger intrinsic fluorescence with quantum yields and lifetimes that depend markedly on solvent polarity and viscosity. It is proposed that 2PE in the red and near-infrared tissue window may allow in situ isomerization of Ecombretastatins to the more active Z-isomer, offering spatial and temporal control of drug activation and constitute a novel form of photodynamic therapy. In the present work we have characterised 2PE of E-CA4 and have used fluorescence lifetime imaging with 2PE to study uptake and intracellular disposition of E-CA4 and an analogue. The results show that these molecules accumulate rapidly in cells and are located mainly in lipidic environments such as lipid droplets. Within the droplets the local concentrations may be up to 2 orders of magnitude higher than that of the drug in the surrounding medium.

Three-dimensional imaging and uptake of the anticancer drug combretastatin in cell spheroids and photoisomerization in gels with multiphoton excitation

“…As in other cells previously studied, 23 uptake of E-combretastatin is fast (t 1∕2 < 2 min) and the compound is mainly located at intracellular loci that have been identified as membranes and lipid droplets. 23 The fluorescence lifetime of the intracellular fluorescence has a distribution that peaks at ca. 1000 ps, longer than in solution (e.g., 280 ps in ethanol and 860 ps in hexane), resulting from a viscous lipidic environment.…”

“…23,24 Uptake and distribution of E-combretastatin A4 (E-CA4) in cell monolayers of melanoma C8161 cells has been studied by real-time FLIM. As in other cells previously studied, 23 uptake of E-combretastatin is fast (t 1∕2 < 2 min) and the compound is mainly located at intracellular loci that have been identified as membranes and lipid droplets. 23 The fluorescence lifetime of the intracellular fluorescence has a distribution that peaks at ca.…”

“…[23][24][25] While Z-combretastatins are nonfluorescent, the intrinsic fluorescence of E-combretastatins has enabled real-time monitoring of E-combretastatin uptake in monolayers of live mammalian cells by two-photon excitation fluorescence lifetime imaging (2PE-FLIM). 23,24 E-combretastatins have fluorescence lifetimes that are dependent on solvent polarity and viscosity and were shown to be proportional to fluorescence quantum yields. Through measurement of both fluorescence intensity and lifetimes, it was possible to evaluate intracellular concentrations.…”

New Approaches to Photodynamic Therapy from Types I, II and III to Type IV Using One or More Photons

“…Unfortunately the combretastatin phosphate salt displayed severe side effects such as cardiovascular toxicity that prevented the compound from making its way to the clinic [118,119]. The E-combretastatin pro-drug uptake and distribution in live cells can be monitored by multiphoton FLIM imaging at 625 nm [120]. It was shown that Ecombretastatins accumulate rapidly in the cytoplasm of live CHO, HeLa and HUVEC (human umbilical vein endothelial cells) cells and particularly in lipidic environments such as lipid droplets at concentrations up to two orders of magnitude higher than that of the pro-drug in the surrounding medium [120,121].…”

“…The E-combretastatin pro-drug uptake and distribution in live cells can be monitored by multiphoton FLIM imaging at 625 nm [120]. It was shown that Ecombretastatins accumulate rapidly in the cytoplasm of live CHO, HeLa and HUVEC (human umbilical vein endothelial cells) cells and particularly in lipidic environments such as lipid droplets at concentrations up to two orders of magnitude higher than that of the pro-drug in the surrounding medium [120,121]. Mechanistically upon excitation of E-combretastatin the molecule is promoted to Figure 7 Type IV direct combretastatin activation by two-photon excitation.…”