X-ray tomography of cryopreserved human prostate cancer cells: mitochondrial targeting by an organoiridium photosensitiser

Abstract

The organoiridium complex Ir[(C,N)₂(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (¹O₂) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of ¹O₂ generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action.

Keywords: Anticancer metallodrugs; Cryo-soft X-ray tomography; Iridium complexes; Phosphorescence; Photodynamic therapy; X-ray microscopy.

Fig. 1

Structure of [Ir(C,N)₂(O,O)] [1] (where C,N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate) [1]

Fig. 2

Cryo-SXT images of cryogenically-fixed PC3 prostate cancer cells grown on carbon–gold TEM grids (24 h) and treated with 1 µM of 1 for 2 h, followed by 10 min irradiation with blue light (λ = 465 nm) and 24 h recovery in complex-free medium: a X-ray mosaic image of a 100 × 100 μm² area of the grid; b X-ray projection image collected from a 16 × 16 μm² area of interest (marked as a red square) in a after exposure to soft x-rays (8 s, 500 eV); c 2D projection of the reconstructed tomogram obtained from the same area (in b); showing 1 = mitochondria, 2 = nucleus, 3 = nuclear membrane, 4 = plasma membrane, 5 = vesicles; d 3D segmented tomogram analysed in SuRVoS and visualised in Amira; showing mitochondria (orange) and nucleus (green). Images were generated using ImageJ (a) [40], IMOD (b, c) [33, 34] and SuRVoS and Amira imaging softwares (d) [34, 36]

Fig. 3

2D projections of reconstructed X-ray tomograms (16 × 16 μm²) of cryogenically fixed PC3 human prostate carcinoma cells grown on Quantifoil TEM grids showing changes in cellular and organelle morphology upon treatment with 1, followed by irradiation with blue light. a Untreated cells in dark conditions (not irradiated with blue light; 465 nm). b Untreated cells exposed to blue light (10 min, 465 nm, 4.8 mW/cm²). c Cells treated with 1 (1 μM) under dark conditions (not irradiated with blue light; 465 nm). d Cells treated with 1 (1 μM) for 2 h, followed by exposure to blue light (10 min, 465 nm, 4.8 mw/cm²). Cellular features shown: 1 = mitochondria, 2 = nucleolus, 3 = nucleus, 4 = nuclear membrane, 5 = features of lamellipodium, 6 = spherical vesicles, 7 = plasma membrane. Images were generated using IMOD software [33, 34]

Fig. 4

Changes in mitochondrial size and structure upon treatment of PC3 cells with 1 followed by irradiation with blue light. a Box and whisker plot showing average volumes (μm³) of segmented mitochondria from cryogenically fixed PC3 prostate carcinoma cells untreated or treated with 1 μM of 1 for 2 h, exposed to 10 min of dark or blue light (465 nm), and followed by 24 h recovery in complex-free medium (37 °C, 5% CO₂). Statistical analysis was performed using Welch’s unpaired t test (assuming unequal variables; *p < 0.05, **p < 0.01, ***p < 0.001). b Representative 2D 4 × 4 μm² sections of X-ray tomograms showing mitochondria from cryogenically fixed PC3 prostate carcinoma cells untreated or treated with 1 μM of 1 for 2 h, exposed to 10 min of darkness or blue light (465 nm), and followed by 24 h recovery in complex-free media (37 °C, 5% CO₂). Images were generated using OriginPro 2018 (a) [50] and IMOD software (b) [33, 34]