Rationally designed ruthenium complexes for 1- and 2-photon photodynamic therapy

Abstract

The use of photodynamic therapy (PDT) against cancer has received increasing attention over recent years. However, the application of the currently approved photosensitizers (PSs) is limited by their poor aqueous solubility, aggregation, photobleaching and slow clearance from the body. To overcome these limitations, there is a need for the development of new classes of PSs with ruthenium(II) polypyridine complexes currently gaining momentum. However, these compounds generally lack significant absorption in the biological spectral window, limiting their application to treat deep-seated or large tumors. To overcome this drawback, ruthenium(II) polypyridine complexes designed in silico with (E,E')-4,4'-bisstyryl-2,2'-bipyridine ligands show impressive 1- and 2-Photon absorption up to a magnitude higher than the ones published so far. While nontoxic in the dark, these compounds are phototoxic in various 2D monolayer cells, 3D multicellular tumor spheroids and are able to eradicate a multiresistant tumor inside a mouse model upon clinically relevant 1-Photon and 2-Photon excitation.

Fig. 1. Chemical structures of complexes 1–7 investigated in this study.

The complexes were isolated as hexafluorophosphate salts.

Fig. 2. Absorption spectra of the complexes 1–7.

a 1P absorption spectrum in acetonitrile and b 2P absorption spectrum in dichloromethane.

Fig. 3. Tumor growth inhibition assay in HeLa MCTS.

Change of the volume in MCTS in correlation to the time of the treatment. The MCTS were treated with compounds 1–7 (20 μM, 2% DMSO, v%), H₂TPP (20 μM, 2% DMSO, v%), and cisplatin (10 μM and 30 μM). The MCTS were a strictly kept in the dark, b exposed to 1P irradiation (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²), c exposed to 2P irradiation (800 nm, 10 J cm⁻² with a section interval of 5 μm) on day 3. The error bars correspond to the standard deviation of the three replicates.

Fig. 4. Representative image of the cell viability assay in HeLa MCTS.

MCTS were treated with compounds 1–7 (20 μM, 2% DMSO, v%) in the dark for 3 days. After this time, MCTS were kept in the dark, exposed to 1P irradiation (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²) or 2P irradiation (800 nm, 10 J cm⁻², and section interval of 5 μm). After 2 days, the cell viability was assessed by measurement of the fluorescence of calcein (λ_ex = 495 nm, λ_em = 515 nm), which is generated in living cells from Calcein AM. The scale bar represents a length of 200 µm.

Fig. 5. Biological Evaluation of 7 inside a mouse model.

In vivo PDT study of 7 using 1P (500 nm, 60 min, 10.0 mW cm⁻², and 36 J cm⁻²) or 2P (800 nm, 50 mW, 1 kHz, pulse width 35 fs, and 5 s mm⁻¹) excitation on nude mice bearing a doxorubicin-selected P-gp-overexpressing human colon cancer tumor (SW620/AD300). a Tumor growth inhibition curves upon treatment. b Average body weights of the tumor-bearing mice. c Representative photographs of the tumor-bearing mice. The error bars correspond to the standard deviation of the five replicates.