Multifunctional Heterometallic IrIII -AuI Probes as Promising Anticancer and Antiangiogenic Agents

Abstract

A new class of emissive cyclometallated Ir^III -Au^I complexes with a bis(diphenylphosphino) methanide bridging ligand was successfully synthesised from the diphosphino complex [Ir(N^C)₂ (dppm)]⁺ (1). The different gold ancillary ligand, a triphenylphosphine (2), a chloride (3) or a thiocytosine (4) did not reveal any significant effect on the photophysical properties, which are mainly due to metal-to-ligand charge-transfer (³ MLCT) transitions based on Ir^III . However, the Au^I fragment, along with the ancillary ligand, seemed crucial for the bioactivity in A549 lung carcinoma cells versus endothelial cells. Both cell types display variable sensitivities to the complexes (IC₅₀ =0.6-3.5 μM). The apoptotic pathway is activated in all cases, and paraptotic cell death seems to take place at initial stages in A549 cells. Species 2-4 showed at least dual lysosomal and mitochondrial biodistribution in A549 cells, with an initial lysosomal localisation and a possible trafficking process between both organelles with time. The bimetallic Ir^III -Au^I complexes disrupted the mitochondrial transmembrane potential in A549 cells and increased reactive oxygen species (ROS) generation and thioredoxin reductase (TrxR) inhibition in comparison with that displayed by the monometallic complex 1. Angiogenic activity assays performed in endothelial cells revealed the promising antimetastatic potential of 1, 2 and 4.

Keywords: anti-angiogenesis; cell imaging; gold; heterometallic compounds; iridium; theranostic agents.

Figure 1

Recent examples of heterometallic theranostic agents. (a), (b), (c).

Scheme 1

Synthesis and chemical structures of complexes 1–4. i) [Ir]: [[Ir(ppy)₂(μ‐Cl)]₂], dppm, MeOH, reflux; ii) [Au(acac)PPh₃], CH₂Cl₂, RT; iii) [AuCl(tht)], Cs₂CO₃, CH₂Cl₂, RT; iv) Cs₂CO₃, thiocytosine, CH₂Cl₂, RT.

Figure 2

Pov‐ray representations of one of the molecules crystallised from complex 2. Counterion, solvent molecules and hydrogens (except hydrogen 24) have been omitted for clarity. The most relevant bond lengths [Å] and angles [°]: Ir1‐C12: 2.063(7), Ir1‐C13: 2.059(7), Ir1‐N1: 2.079(6), Ir1‐N2: 2.068(5), Ir1‐P1: 2.388(5), Ir1‐P2: 2.421(2), Au1‐C24: 2.109(7), Au1‐P3: 2.274(1), C24‐Au1‐P3: 177.9(2), P2‐Ir1‐P1: 68.77(6), N2‐Ir1‐N1: 165.8(6), P1‐C24‐P1: 96.0(3).

Figure 3

Emission spectra of complexes 1–4 measured in DSMO solution.

Figure 4

Cell death mechanism induced by complexes 1 and 4 in A549 cells. Top: cytotoxic effect of compound 4 evaluated with Annexin V‐DY634 and 7‐AAD staining. Bottom: percentage of apoptotic cells when pre‐incubated with 30 μM Z‐VAD‐fmk or not before adding the complex.

Figure 5

Fluorescence confocal microscopy images of A549 cells incubated with 1 (24 h) and stained with MTR. Image after irradiation at A) 473 and B) 598 nm. C) Superimposition of (A) and (B). D) Superimposition with phase contrast. Green: complex 1, red: MTR; image width (↔): 210 μm.

Figure 6

Cross section of intensity for the superimposition of compound 1 (green, irradiation at 473 nm) with MTR (red, irradiation at 598 nm) incubated in A549 cells. Image width (↔): 210 μm.

Figure 7

Fluorescence confocal microscopy images of A549 cells incubated with 2 (top) and 3 (bottom) for 24 h and stained with MTR. Images after irradiation at A) 473 and B) 598 nm. C) Superimposition of (A) and (B). D) Superimposition with phase contrast. Green: complexes 2 and 3, red: MTR; image width (↔): 210 μm.

Figure 8

Fluorescence confocal microscopy images of A549 cells incubated with 1 (18 h) and stained with LTR. Image after irradiation at A) 473 and B) 598 nm. C) Superimposition of (A) and (B). D) Superimposition with phase contrast. Green: complex 1, red: LTR; image width (↔): 210 μm.

Figure 9

Fluorescence confocal microscopy images of A549 cells incubated with 3 (18 h) and stained with LTR. Image after irradiation at A) 473 and B) 598 nm. C) Superimposition of (A) and (B) and cross‐section of their intensities. Green: complex 3, red: LTR; image width (↔): 210 μm.

Figure 10

Fluorescence confocal microscopy images of A549 cells incubated with 4 (2 h) and stained with LTR. Image after irradiation at A) 473 and B) 598 nm. C) Superimposition of (A) and (B). D) Superimposition with phase contrast. Green: complex 4, red: LTR; mage width (↔): 210 μm.

Figure 11

ROS production induced by 1 and 4 in A549 cells.

Figure 12

Inhibition of thioredoxin assay for complexes 1 and 4. Representation of the evolution of the absorbance intensity of TNB over 5 min.

Figure 13

Disruption of mitochondrial transmembrane potential induced by 4. Positive control: S63845.

Figure 14

Phase‐contrast microscopy images of apb‐ECFCs incubated with 1, 2 and 4 at 1 μM for 24 h.

Figure 15

Phase‐contrast microscopy images of the in vitro angiogenic activity assays in ucb‐ECFCs cells incubated with 1, 2 and 4 at 0.25 μM for 20 h.