Photoactivated cell-killing involving a low molecular weight, donor-acceptor diphenylacetylene

Abstract

Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class of photosensitiser, exemplified by DC324, has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of in vitro cellular assays confirmed ROS formation and in vivo cytotoxic activity was exemplified via irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.

Fig. 1. Comparison of the characteristics of DC324 and DC473 with those of existing photosensitisers, KillerRed and Photofrin®.

Fig. 2. Synthesis of acceptor alkyne 4.

Fig. 3. Coupling of acceptor alkyne 4 and donor tetrahydroquinoline 5 (ref. 34) to give DC324.

Fig. 4. (A) Absorption spectra of DC324 in a variety of solvents and electron density difference plots for the two observed major absorption bands S₀ → S₁ (low energy) and S₀ → S₂ (high energy) obtained from TD-DFT calculations at the M06-D3-BJ/def2-TZVP level of theory. (B) Emission spectra of DC324 in a variety of solvents. (C) Two-photon absorption spectra of DC324 and DC473 in toluene. (D) HaCaT keratinocytes treated with 10 μM DC324 for 30 min, prior to irradiation. (E) HaCaT keratinocytes treated with 10 μM DC324 after multiple short irradiations with 405 nm, 12 hours after initial imaging.

Fig. 5. (A) HaCaT keratinocytes treated with 1 μM DC324 (green) and co-stained with MitoTracker Red (red). Scale bars equal 50 μm. (B) Peak emission wavelength of DC324 in regions of interest (ROI) of cells, with excitation at 405 nm. (C) ROI 1, green circle marks area distal to nuclear membrane (λ_max = 490 nm) and ROI 2, blue circle, marks area adjacent to nucleus (λ_max = 460 nm). Average emission wavelength for red square was calculated (Average ROI λ_max = 475 nm).

Fig. 6. Detection of fluorescence produced by ROS reactive dye, CellRox in HaCaT keratinocytes treated with 1 μM DC324 or a DMSO control. Scale bars equal 25 μm.

Fig. 7. (A) Chemical structure of synthetic retinoid, EC23. (B) Quantitation of relative fluorescence of CellRox before and after irradiation using a 405 nm laser. The relative fluorescence intensity was calculated in the region of interest (ROI, yellow box) at each time point and data graphed using box-whisker plot. Graph coloured lines denote sample mean (n = 11, DC324; n = 3, EC23; n = 3, negative) from 2 experimental replicates for each treatment (red = DC324, pink = EC23, blue = negative). Time 0 equals time of irradiation. Increased fluorescence intensity in DC324-treated cells was significant as determined by one-way ANOVA (p < 0.001). Scale bars equal 50 μm.

Fig. 8. (A) Phase contrast image of confluent sheet of HaCaT epithelial cells exposed to 10 μM DC324 and the righthand area of the field irradiated with UV-A. Image captured 10 minutes post irradiation; note that cell death occurred only in the irradiated zone. Scale bars equal 100 μm. (B) Cell viability after irradiation with varying UV-A doses. Dose–response curves have been fitted to the data in a box-whisker plot graph using 4 experimental replicates (n = 4). Statistical significance was determined by one-way ANOVA (p < 0.0001) and Dunnett's multiple comparison test comparing individual dose response curves to unirradiated controls (0 J cm^–2). The energy fluence of 14, 28 and 42 J cm^–2 were considered statistically significant with p values of 0.0034, 0.0002 and <0.0001, respectively. (C) The average viable fraction of HaCaT epithelial cellular populations 24 hours after an initial 60 second UV-exposure, as a function of the concentration of compound administered. A non-linear regression curve is fitted to the data (experimental replicates, n = 3; R² = 0.99).

Fig. 9. 48 hour wild-type zebrafish embryo treated with 1 μM DC324 for 2 hours prior and the tail irradiated with 42 J cm^–2 of UV-A (time = 0 minutes); phase contrast images captured at the time of irradiation (A/B) and 40 minutes after irradiation (C).