Dual Emissive Ir(III) Complexes for Photodynamic Therapy and Bioimaging

Abstract

Photodynamic therapy (PDT) is a cancer treatment still bearing enormous prospects of improvement. Within the toolbox of PDT, developing photosensitizers (PSs) that can specifically reach tumor cells and promote the generation of high concentration of reactive oxygen species (ROS) is a constant research goal. Mitochondria is known as a highly appealing target for PSs, thus being able to assess the biodistribution of the PSs prior to its light activation would be crucial for therapeutic maximization. Bifunctional Ir(III) complexes of the type [Ir(C^N)₂(N^N-R)]⁺, where N^C is either phenylpyridine (ppy) or benzoquinoline (bzq), N^N is 2,2'-dipyridylamine (dpa) and R either anthracene (1 and 3) or acridine (2 and 4), have been developed as novel trackable PSs agents. Activation of the tracking or therapeutic function could be achieved specifically by irradiating the complex with a different light wavelength (405 nm vs. 470 nm respectively). Only complex 4 ([Ir(bzq)₂(dpa-acr)]⁺) clearly showed dual emissive pattern, acridine based emission between 407-450 nm vs. Ir(III) based emission between 521 and 547 nm. The sensitivity of A549 lung cancer cells to 4 evidenced the importance of involving the metal center within the activation process of the PS, reaching values of photosensitivity over 110 times higher than in dark conditions. Moreover, complex 4 promoted apoptotic cell death and possibly the paraptotic pathway, as well as higher ROS generation under irradiation than in dark conditions. Complexes 2-4 accumulated in the mitochondria but species 2 and 4 also localizes in other subcellular organelles.

Keywords: cytotoxicity; dual emiter; fluorescence microscopy; iridium; mitochondria; optical properties; photodynamic therapy.

Figure 1

Chemical structures of Photofrin, TLD1433, and complexes (A,B) adapted from [8,18,22,23].

Figure 2

Depiction of the devised dual emissive Ir(III) PSs.

Scheme 1

Synthetic pathway for the synthesis of L1 and L2 and complexes 1–4. Reaction conditions as follow: (i) ^tBuOK, toluene, reflux, 12 h; (ii) MeOH, reflux, 15 h under Ar.

Figure 3

Emission spectra of complexes 1–4 in DMSO solution at 298 K.

Figure 4

Excitation spectra (left) of complex 4 for the different emission profiles and the correspondent emission spectra (right) upon excitation at different wavelengths.

Figure 5

Phase contrast microscopy images of A549 cells treated with L1, L2 and 1–4 at concentrations between 10 and 20 µM for 20 h. Black arrows point to apoptotic cells. Little black triangles show examples of cells containing cytoplasmic vesicles.

Figure 6

Cytotoxicity assays of complex 4, when it was incubated in A549 cell line for 24 h, in a range of concentration between its IC₅₀ and ¼ IC₅₀ values. In dark (left); Under irradiation at 470 nm (right).

Figure 7

Cell cycle analysis after treatment with complex 4 in dark (top graphs) and under irradiation at 470 nm (bottom graphs). The cell distribution at each phase is indicated for each graphic.

Figure 8

Fluorescence confocal microscopy images in A549 cells incubated with complexes 2, 3, 4 (green, irradiated at 458 nm) and stained with MTR (red, irradiation at 598 nm).

Figure 9

Superimposition images in A549 cells of complexes 2 (A) and 4 (B) incubated 2 h and stained with MitoTracker Red (MTR).

Figure 10

Generation of ROS induced by complex 4 at increasing concentrations in dark (left graph) and upon irradiation at 470 nm (right graph).