Oxygen Mapping of Melanoma Spheroids using Small Molecule Platinum Probe and Phosphorescence Lifetime Imaging Microscopy.

Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application.

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

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.

Synthesis and photophysical properties of Ir(iii)/Re(i) dyads: control of Ir→Re photoinduced energy transfer.

A series of dinuclear Ir(iii)/Re(i) complexes has been prepared based on a family of symmetrical bridging ligands containing two bidentate N,N'-chelating pyrazolyl-pyridine termini, connected by a central aromatic or aliphatic spacer. The Ir(iii) termini are based on {Ir(F2ppy)2}(+) units (where F2ppy is the cyclometallating anion of a fluorinated phenylpyridine) and the Re(i) termini are based on {Re(CO)3Cl} units. Both types of terminus are luminescent, with the Ir-based unit showing characteristic strong, structured phosphorescence in the blue region (maximum 452 nm) with a triplet excited state energy of 22 200 cm(-1) and the Re-based unit showing much weaker and lower-energy phosphorescence (maximum 530 nm) with a triplet excited state energy of 21 300 cm(-1). The energy gradient between the two excited states allows for partial Ir→Re photoinduced energy-transfer, with substantial (but incomplete) quenching of the higher-energy Ir-based emission component and sensitised emission - evidenced by an obvious grow-in component - on the lower-energy Re-based emission. The Ir→Re energy-transfer rate constants vary over the range 1-8 × 10(7) s(-1) depending on the bridging ligand: there is no simple correlation between bridging ligand structure and energy-transfer rate, possibly because this will depend substantially on the conformation of these flexible molecules in solution. To test the role of ligand conformation further, we investigated a complex in which the bridging chain is a (CH2CH2O)6 unit whose conformation is known to be solvent-polarity dependent, with such chains adopting an open, elongated conformation in water and more compact, folded conformations in organic solvents. There was a clear link between the rate and extent of Ir→Re energy-transfer which reduced in polar solvents as the chain became elongated and the Ir/Re separation was larger; and increased in less polar solvents as the chain adopted a more compact conformation and the Ir/Re separation was reduced.

Heteronuclear Ir(III)-Ln(III) Luminescent Complexes: Small-Molecule Probes for Dual Modal Imaging and Oxygen Sensing.

Luminescent, mixed metal d-f complexes have the potential to be used for dual (magnetic resonance imaging (MRI) and luminescence) in vivo imaging. Here, we present dinuclear and trinuclear d-f complexes, comprising a rigid framework linking a luminescent Ir center to one (Ir·Ln) or two (Ir·Ln2) lanthanide metal centers (where Ln = Eu(III) and Gd(III), respectively). A range of physical, spectroscopic, and imaging-based properties including relaxivity arising from the Gd(III) units and the occurrence of Ir(III) → Eu(III) photoinduced energy-transfer are presented. The rigidity imposed by the ligand facilitates high relaxivities for the Gd(III) complexes, while the luminescence from the Ir(III) and Eu(III) centers provide luminescence imaging capabilities. Dinuclear (Ir·Ln) complexes performed best in cellular studies, exhibiting good solubility in aqueous solutions, low toxicity after 4 and 18 h, respectively, and punctate lysosomal staining. We also demonstrate the first example of oxygen sensing in fixed cells using the dyad Ir·Gd, via two-photon phosphorescence lifetime imaging (PLIM).

Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand.

Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1-1 µM) by comparatively low dose of 405 nm light (3.6 J cm(-2)) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.

Porphyrin/Platinum(II) C

A new class of substituted porphyrins has been developed in which a different number of cyclometalated Pt(II) C^N^N acetylides and polyethylene glycol (PEG) chains are attached to the meso positions of the porphyrin core, which are meant for photophysical, electrochemical, and in vitro light-induced singlet oxygen ((1)O2) generation studies. All of these Zn(II) porphyrin-Pt(II) C^N^N acetylide conjugates show moderate to high (ΦΔ =0.55 to 0.63) singlet oxygen generation efficiency. The complexes are soluble in organic solvents but, despite the PEG substituents, slowly aggregate in aqueous solvent systems. These conjugates also exhibit interesting photophysical properties, including near-complete photoinduced energy transfer (PEnT) through the rigid acetylenic bond(s) from the Pt(II) C^N^N antenna units to the Zn(II) porphyrin core, which shows sensitized luminescence, as shown by quenching of Pt(II) C^N^N-based luminescence. Electrochemical measurements show a set of redox processes that are approximately the sum of what is observed for the Pt(II) C^N^N acetylide and Zn(II) porphyrin units. UV/Vis spectroscopic properties are supported by DFT calculations.

A new ligand skeleton for imaging applications with d-f complexes: combined lifetime imaging and high relaxivity in an Ir/Gd dyad.

A new rigid and conjugated ligand structure connecting phenanthroline and poly(amino-carboxylate) binding sites provides d-f complexes which show high potential for use in dual (luminescence + magnetic resonance) imaging and for optimisation of d → f photoinduced energy-transfer.

Combined two-photon excitation and d→f energy transfer in a water-soluble Ir(III)/Eu(III) dyad: two luminescence components from one molecule for cellular imaging.

The first example of cell imaging using two independent emission components from a dinuclear d/f complex is reported. A water-stable, cell-permeable Ir(III) /Eu(III) dyad undergoes partial Ir→Eu energy transfer following two-photon excitation of the Ir unit at 780 nm. Excitation in the near-IR region generated simultaneously green Ir-based emission and red Eu-based emission from the same probe. The orders-of-magnitude difference in their timescales (Ir ca. µs; Eu ca. 0.5 ms) allowed them to be identified by time-gated detection. Phosphorescence lifetime imaging microscopy (PLIM) allowed the lifetime of the Ir-based emission to be measured in different parts of the cell. At the same time, the cells are simultaneously imaged by using the Eu-based emission component at longer timescales. This new approach to cellular imaging by using dual d/f emitters should therefore enable autofluorescence-free sensing of two different analytes, independently, simultaneously and in the same regions of a cell.

Dinuclear ruthenium(II) complexes as two-photon, time-resolved emission microscopy probes for cellular DNA.

The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging.