2.1.

Synthesis of Combretastatin Derivatives

$E$- and $Z$-isomers of CA4 (CA4, 1-(3′,4′,5′-trimethoxyphenyl)-2-(4″-methoxy-3″-hydroxy-phenyl)ethene) and CA4F (1-(3′,4′,5′-trimethoxyphenyl)-2-(3″-fluoro-4″-methoxyphenyl)ethene) were synthesized as previously described.^9,16 Structures and purity were determined by TLC and ${}^1\mathrm{H}$ and ${}^{13}\mathrm{C}$ NMR.

2.2.

Confocal and Multiphoton FLIM

Confocal images, two-photon excited FLIM images and two-photon emission spectra used a combined laser scanning confocal multiphoton FLIM and spectral detection apparatus based on a Nikon TE2000-U inverted fluorescence microscope^3,17 with a $60\times$ water immersion objective (numerical aperture 1.2). For lifetime and FLIM measurements with $E$-CA4 and $E$-CA4F, BG3 (Comar) and a narrowband interference (400IU25) filters were used to isolate the light transmitted to the photomultiplier. Excitation for 2PE at 625 nm was provided by a titanium-sapphire laser (Mira, Coherent Ltd., Santa Clara, California; 180-fs laser pulses at 76 MHz) pumping an optical parametric oscillator (Coherent Ltd.). Samples were irradiated on the motorized and temperature controlled (Peltier heater/cooler) microscope stage. Confocal imaging using 488- and 543-nm excitation was carried out using Nikon eC1-Si or eC2 confocal laser scanning accessories mounted on the same Nikon microscope.

2.3.

One-Photon Photoisomerization Quantum Yield Measurements

Spectroscopic grade solvents (Sigma-Aldrich, Gillingham, United Kingdom and Alfa Aesar, Heysham, United Kingdom) were used as supplied. Aberchrome 540 was used for actinometry.¹⁸ UV–visible spectra were measured using a PerkinElmer Lambda 25. Steady-state fluorescence emission measurements were recorded using a Horiba FluoroMax-3 using the manufacturer supplied spectral correction curves. Irradiations were carried out within the fluorimeter sample cell with a 5-nm excitation slit width. Combretastatin solutions in a 1 cm quartz cuvette were deaerated by nitrogen bubbling inside an AtmosBag (Sigma-Aldrich) to minimize phenanthrene formation. Quantum yields ($\mathrm{\Phi }$) for the $E\rightleftharpoons Z$-isomerization of the different combretastatins were determined from the photon flux and the initial changes in absorbance.¹⁹ The concentrations of $E$- and $Z$-isomers ($c^E$ and $c^Z$) at the photostationary state were calculated using extinction coefficients (${\epsilon }^E$ and ${\epsilon }^Z$) and quantum yields derived from the kinetic measurements [Eq. (1)], and were found to be consistent with experimental measurements at the establishment of the photostationary state.¹⁰

2.4.

Cell Culture

Chinese hamster ovary (CHO) cells were obtained from the European Collection of Cell Cultures and were grown and maintained in phenol-red free Dulbecco’s modified Eagle medium (Gibco, Paisley, United Kingdom) and minimal essential medium (Gibco), respectively, supplemented with foetal calf serum (FCS; 10%), penicillin ($100\mathrm{U}/\mathrm{ml}$), streptomycin ($100\mu \text{g}/\mathrm{ml}$), and l-glutamine (2 mM). Cells were seeded at densities of $2\times {10}^5$ cells/dish on MatTek glass-bottom culture dishes (35 mm $\varnothing$, No. 1.5, uncoated, $\gamma$-irradiated) (MatTek Corporation, Ashland, Massachusetts) and placed in an incubator under a humidified atmosphere (37°C, 5% ${\mathrm{CO}}_2$) for 24 h to adhere.

2.5.

Combretastatin Induced Apoptosis Monitored by Confocal Imaging of Annexin V AlexaFluor488 Conjugate and Propidium Iodide Staining on Live Cells

Combretastatin induced apoptosis on CHO cell monolayers was determined using annexin V AlexaFluor 488 conjugate (Invitrogen, Paisley, United Kingdom) as an indicator for the loss of phospholipid asymmetry in the plasma membrane of apoptotic cells in the presence of ${\mathrm{Ca}}^{2+}$ (2.5 mM), and propidium iodide (PI) as a DNA intercalating fluorescent dye that is permeable to membranes of apoptotic or necrotic cells. The cytotoxicity of $Z$-combretastatins in the CHO cell monolayers was induced by addition of aliquots of a stock solution in dimethyl sulfoxide (DMSO) ($\le 5\%$). Following incubation for 48 h, the tissue culture medium was aspirated and replaced with ${\mathrm{CaCl}}_2$ and HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] supplemented medium. The annexin V conjugate was added at 0.5% final concentration ($5\mu \mathrm{l}/\mathrm{ml}$ medium). A PI stock solution ($1.6\mu \mathrm{l}/\mathrm{ml}$) was added, and the samples incubated for 15 min in the dark and imaged using confocal laser scanning microscopy (excitation at 543 nm for PI and 488 nm for annexin V conjugate). For irradiations in the presence of $E$-combretastatins, a $100\times 100\mu \mathrm{m}$ field was exposed to 625-nm light by raster-scanning the multiphoton beam for 10 min allowing a total of 20 scans with a pixel dwell time of 2 ms. Samples were incubated for 24 h and examined for apoptosis as described above.

3.1.

Photoisomerization Induced by One- and Two-Photon Excitations In Vitro

$E\rightleftharpoons Z$ photoisomerization of $E$-combretastatin A4 ($E$-CA4) (1 mM) in deaerated methanol on irradiation at 340 nm is illustrated in Fig. 2 through changes in absorption spectra. As the irradiation progresses, absorbance of the $E$-isomer at ${\lambda }_{\max }$ (329 nm) decreases and is replaced by that of the $Z$-isomer with ${\lambda }_{\max }$ 287 nm with a lower extinction coefficient. The spectra show good isosbestic points showing that only the two species are involved and that phenanthrene formation was effectively suppressed by deaeration. A plot of A (340 nm; inset to Fig. 2) indicates progression to the stationary state mixture of $E$- and $Z$-isomers. Quantum yields for photoisomerization were calculated from both the initial rate of absorbance change and the absorbance at the stationary state as detailed in Sec. 2, and are shown in Table 1 for CA4 and CA4F in methanol solution. The values show that photoisomerization is relatively efficient with quantum yields in the range of 0.27 to 0.48 and are comparable with published data for a wide range of other stilbenes in fluid solution at room temperature.¹⁰

Fig. 2

Changes in the absorption spectrum of $E$-CA4 ($67\mu \mathrm{M}$) in $N_2$-saturated methanol on irradiation at 340 nm. The inset shows the change in absorbance at 340 nm with irradiation time.

Table 1

Quantum yields for photoisomerization of E- and Z-isomers of CA4 and CA4F in methanol solution. The E/Z ratios at the photostationary state were measured experimentally based on the extinction coefficients and compared with those calculated according to Eq. (1).

Photoisomerization of combretastatins by 2PE was inferred from measurements of fluorescence intensity versus laser power on irradiation at 624 nm in the microscope system using femtosecond laser pulses. Under these conditions, 2PE excitation takes place within the femtoliter focal volume, from which the product diffuses into bulk solution, away from the detectable zone, resulting in an equilibrium concentration balancing formation and diffusion. The fluorescence intensity ($F$) from a solution of $E$-CA4F versus laser power ($P$; inset to Fig. 3) shows the expected quadratic relationship ($F\propto P^n$ with $n=2.07\pm 0.05$) at low laser powers up to about 1.5 mW. However, a plot of $F/P$ (Fig. 3) shows deviation from the expected linear behavior because of saturation at higher laser powers that is indicative of depletion of the ground state $E$-CA4F molecules as a result of photoisomerization. The saturation threshold of $\sim 2\mathrm{mW}$ laser power is less than that expected ($\sim 30\mathrm{mW}$) from simple excitation and fluorescence decay²⁰ and strongly suggests that the observed saturation results from conversion of the fluorescent $E$-isomer to the nonfluorescent $Z$-isomer by photoisomerization. For the nonfluorescent $Z$-combretastatins, fluorescence was observed as the laser power was increased and is interpreted as arising from photoisomerization to the fluorescent $E$-isomer. Through a series of calibrations using solutions of increasing concentrations of $E$-combretastatin, the photoisomerization could be quantified and shown to produce up to 25% conversion within the focal volume at an average laser power of 4.5 mW (Fig. 4). These measurements probe only the focal volume, from which photochemical products will rapidly diffuse, and it is concluded that in solution sufficient amounts of $Z$-combretastatins are formed by 2PE photoisomerization of $E$-combretastatins to exert a cytotoxic effect.

Fig. 3

Effect of average laser power ($P$) on fluorescence intensity ($F$, at 390 nm) from a solution of $E$-CA4F (0.5 mM) in ethanol on illumination with the femtosecond laser beam at 624 nm. The main figure shows a plot of $F/P$ versus $P$ with the solid line showing the expected linear plot for two-photon excitation. The inset shows a direct plot of $F$ versus $P$ data showing the expected quadratic relation at low laser powers confirming the 2PE process under these conditions.

Fig. 4

Percentage conversions to the $E$-isomers deduced from fluorescence intensities produced by a focused laser beam excitation at 590 nm in 1 mM DMSO solutions of $Z$-CA4 (closed square) and $Z$-CA4F (open square).

3.2.

Cell Death Induced by Combretastatin Two-Photon Isomerization

$Z$-CA4 induced apoptosis has been reported in human endothelial cells.²¹ Staining for cell death (PI, red fluorescence) and apoptosis (annexin V AlexaFluor488 conjugate, green fluorescence) in cultured CHO cells following exposure to $Z$-CA4F [Fig. 5(A)] shows significant apoptosis at $1\mu \mathrm{M}$ drug. The combined effects of laser irradiation at 625 nm and exposure to $E$-CA4 on live cell CHO monolayers are shown in Fig. 5(B). The central $100{\mu \text{m}}^2$ area of each field was illuminated by raster scanning a focused laser beam (625 nm). PI and annexin V staining reveal the effects of increasing laser power and $E$-CA4 concentration. Control cells [no $E$-CA4, Fig. 5(B), panels a1–e1] show only occasional staining for cell damage at low laser powers ($<6\mathrm{mW}$), but at the higher powers ($>6\mathrm{mW}$) there are signs of light-induced cell damage in the central illuminated region. Cells subjected to combined incubation with $E$-CA4 and laser illumination display positive staining for cell death within the laser exposed central regions of the fields shown, whereas the unexposed peripheral areas remain largely unaffected. These unirradiated border regions represent the control of cells in the presence of the drug without light. At $E$-CA4 concentrations of $25\mu \mathrm{M}$ and below, these peripheral areas show little cell damage, emphasizing the difference in toxicity between the $E$-isomer [Fig. 5(B)] and the $Z$-isomer [Fig. 5(A)]. Optimal induction of cell damage by combined effects of light and $E$-CA4 appears at 4.7 mW laser power and $10\mu \mathrm{M}$ $E$-CA4 (panel c2). It is noticeable that even in the presence of higher concentrations of $E$-CA4, the highest laser power damages the cells to such an extent that they show less overall staining with both labels (e1–e4). Similar results were obtained with $E$-CA4F.

Fig. 5

Apoptosis and cell permeability induced in CHO cell monolayers by combretastatins assessed by staining with annexin V AlexaFluor488 conjugate (green) and propidium iodide (red), respectively. (A) Effect of increasing concentration of $Z$-CA4F (0, 0.1, and $1\mu \mathrm{M}$) after 48 h. (B) Effects of $E$-CA4 (0, 10, 25, and $50\mu \mathrm{M}$) added 2 h before irradiation of a $100\times 100\mu \mathrm{m}$ field for 10 min at 625 nm with increasing laser powers at the sample (1.6, 3.1, 4.7, 6.3, and 9.4 mW).