Metal Complexes for Two‐Photon Photodynamic Therapy: A Cyclometallated Iridium Complex Induces Two‐Photon Photosensitization of Cancer Cells under Near‐IR Light

Abstract Photodynamic therapy (PDT) uses photosensitizers (PS) which only become cytotoxic upon light‐irradiation. Transition‐metal complexes are highly promising PS due to long excited‐state lifetimes, and high photo‐stabilities. However, these complexes usually absorb higher‐energy UV/Vis light, whereas the optimal tissue transparency is in the lower‐energy NIR region. Two‐photon excitation (TPE) can overcome this dichotomy, with simultaneous absorption of two lower‐energy NIR‐photons populating the same PS‐active excited state as one higher‐energy photon. We introduce two low‐molecular weight, long‐lived and photo‐stable iridium complexes of the [Ir(N^C)2(N^N)]+ family with high TP‐absorption, which localise to mitochondria and lysosomal structures in live cells. The compounds are efficient PS under 1‐photon irradiation (405 nm) resulting in apoptotic cell death in diverse cancer cell lines at low light doses (3.6 J cm−2), low concentrations, and photo‐indexes greater than 555. Remarkably 1 also displays high PS activity killing cancer cells under NIR two‐photon excitation (760 nm), which along with its photo‐stability indicates potential future clinical application.

. Photophysical data for complexes 1
Chemical shift data are in ppm; spectra are referenced using residual solvent 1 H and 13 C shifts. 1 H coupling constants J are reported in Hz to the nearest 0.5 Hz. Scans of the 1 H NMR spectra of complexes 1 and 2 are provided in the Supplementary Information. High resolution mass spectrometry was carried out using electrospray ionisation on a Waters LCT Premier XE or a Thermo-Finnigan LTQ FT mass spectrometer 1,1'-Dimethyl-2,2'-bisbenzimidazole 2,2'-Bisbenzimidazole (50 mg, 0.213 mmol) and potassium carbonate (74 mg, 0.533 mmol) were stirred in DMF (5 mL) for 30 min, and iodomethane (181 mg, 1.28 mmol) was then added. The mixture was stirred for 12 h. The beige precipitate that had formed was separated by centrifugation, washed with diethyl ether (3 × 10 mL) and acetone (2 × 10 ml), and dried to give the desired product (42 mg, 75%). M.p. > 250˚C. 1    obtained by applying a linear fit. The gradient (b) was then used to calculate the twophoton absorption cross-section according equation (1) reported in [2] .
Where σ is the two-photon absorption cross-section, b is the slope of linear dependence of F vs. W 2 , c is the molar concentration, and φ is the differential emission quantum yield in the 5nm spectral range selected for comparison. The subscript s or r means either sample or reference. The value σ r for Fluorescein was taken as 45 GM at 760 nm.
The relative quantum yield φ was obtained on a Jobin-Yvon Fluoromax 4 fluorimeter under one-photon excitation. The total emission quantum yield was measured for aerated solutions of complex 1 in the appropriate solvents, using [Ru(bipy )3 ]Cl 2 (ϕ = 0.028 [3] ) as the reference standard. The relative emission quantum yield φ was calculated by multiplying the total quantum yield by the fraction of emission intensity in the 5 nm range previously selected, with the respect to the intensity of entire emission spectrum.

Cell culture
The

Colocalisation studies
Cover glasses (22 x 22 mm) were sterilised (industrial methylated spirits, IMS) and placed flat in 6 well plates. Cells were seeded at a density of ~1 × 10 4 cells per well and allowed to adhere overnight in culture media. Complex 1 (1 µM) was added and incubated (2 hours). Co-stains were added as follows:

MitoTracker ® Orange
After Inhibitor assay [4] Cover glasses (22 Plates were incubated for 5-10 days until colonies formed and stained as above.

Singlet oxygen measurements
Due to the short lifetime of singlet oxygen in an aqueous environment, singlet oxygen was detected directly in dichloromethane (DCM) by measurement of singlet oxygen luminescence(λ em ~1275 nm) following photo excitation of complexes 1 and 2 at room temperature in air saturated solutions of DCM [5] . The quantum yield of singlet oxygen production was determined by comparing the slopes of the linear plots of the initial intensity vs. laser energy for the compounds and that of the standard (perinapthenone, φ( 1 O 2 )=95%) [6] . Emission lifetime for 1 Δ g sensitised by the compounds and the standard were similar (within the range 70-90 µs), indicating that 1 Δ g does not react with the photosensitiser in its ground state.

Reactive oxygen species detection
The generation of reactive oxygen species, ROS, was monitored using the

Apoptosis/cell death assay
Cells were plated on cell culture dishes at 1x10 6 cells/plate and incubated overnight.
Plates were then incubated with 0.1-1 µM complex 1 or DMSO control for 2 hours before light treatment following the photodynamic activity assay. Following light treatment, cells were washed once with PBS and cell pellets stained for annexin V using Flowcollect ® Annexin Red Kit (Millipore (U.K.) Limited, Watford, UK) according to manufactures instructions. Analysis was performed by flow cytometry (BD™ LSR II flow cytometer).

Photodynamic activity assay (two-photon)
A 96-well plate (Ibidi, Thistle Scientific LTD, Glasgow, UK) was seeded and incubated overnight to give a seeding density of ~80%. Wells were treated with 1 µM