Exploring Structure-Activity Relationships in Photodynamic Therapy Anticancer Agents Based on Ir(III)-COUPY Conjugates

Abstract

Photodynamic therapy holds great promise as a non-invasive anticancer tool against drug-resistant cancers. However, highly effective, non-toxic, and reliable photosensitizers with operability under hypoxic conditions remain to be developed. Herein, we took the advantageous properties of COUPY fluorophores and cyclometalated Ir(III) complexes to develop novel PDT agents based on Ir(III)-COUPY conjugates with the aim of exploring structure-activity relationships. The structural modifications carried out within the coumarin scaffold had a strong impact on the photophysical properties and cellular uptake of the conjugates. All Ir(III)-COUPY conjugates exhibited high phototoxicity under green light irradiation, which was attributed to the photogeneration of ROS, while remaining non-toxic in the dark. Among them, two hit conjugates showed excellent phototherapeutic indexes in cisplatin-resistant A2780cis cancer cells, both in normoxia and in hypoxia, suggesting that photoactive therapy approaches based on the conjugation of far-red/NIR-emitting COUPY dyes and transition metal complexes could effectively tackle in vitro acquired resistance to cisplatin.

Figure 1

(A) Structure of previously evaluated compounds: COUPY coumarin 1a, cyclometalated Ir(III) complex 2a, and the corresponding Ir(III)-COUPY conjugate 3a. (B) Structure of the new COUPY coumarins (1b–1c) and Ir(III)-COUPY conjugates (3b–3e) investigated in this work.

Scheme 1. Synthesis of Ir(III)-COUPY Conjugate 3b and of Control COUPY Coumarin 1b

Scheme 2. Synthesis of Ir(III)-COUPY Conjugate 3c and Control COUPY Coumarin 1c

Scheme 3. Synthesis of Ir(III)-COUPY Conjugates 3d and 3e

Figure 2

Comparison of the molar absorptivity (a) and emission spectra (b) of Ir(III)-COUPY conjugates in ACN.

Figure 3

Detection of ROS in solution. (a) Increase of the fluorescence spectra emission of DHR123 upon photoirradiation of COUPY coumarin 1a, Ir(III) complex 2a, Ir(III)-COUPY conjugates 3a, 3b, and 3c or without any compound (DHR 123 alone) at 505 nm in PBS (0.2% DMSO). DHR123 fluorescence was excited at 500 nm. (b) EPR spectra of Ir(III)-COUPY conjugates 3a–3c trapped by DMPO or TEMP in MeOH in the dark and after 5 min of irradiation with green light (530 nm and 450 mW cm^–2).

Figure 4

Cellular uptake and photocytotoxicity of Ir-COUPY conjugates in cancer cells. (a) Intracellular accumulation of Ir(III) compounds and cisplatin in A2780cis cells after 2 h treatment at 10 μM. Data expressed as mean ± SD from three independent measurements. (b) Single confocal planes of HeLa cells incubated with the compounds for 30 min at 37 °C. Upper row: Merge of bright field and fluorescence images. Lower row: Fluorescence images of the compounds. Scale bar: 20 μm. (c–d) Summary of in vitro photocytotoxicity for compounds 1a–1c, 2a, and 3a–3e in A2780cis cells after light irradiation (520 nm, 1.5 mW cm^–2, 1 h) under normoxia (21% O₂) or hypoxia (2% O₂) represented as log(IC₅₀^LIGHT) and phototherapeutic indexes (PI), defined as the ratio of dark to light IC₅₀ values. IC₅₀, PI values, and their corresponding SD errors are listed in the Supporting Information (Table S3).

Figure 5

Photoinduced cell death mechanism of Ir-COUPY conjugates. (a) Relative ROS levels in A2780cis cells under normoxia (21% O₂) and hypoxia (2% O₂) after treatment with the compounds at 10 μM in dark or upon light irradiation (520 nm, 1.5 mW cm^–2, 1 h) as measured with the DCFH-DA probe. Menadione (Men, 50 μM) was used as a positive control for ROS induction. Data expressed as mean ± SD from three independent measurements. (b) Cell death induction in A2780cis after PDT treatment with Ir(III)-COUPY agents. Data from three independent flow cytometry experiments using Annexin V/PI dual staining. (c) Change in the relative volume of A2780cis MCTS over 7 days. PDT treatments were applied on day 3 (dashed line). Error bars correspond to SD from three replicates. Statistical significance *p < 0.05, **p < 0.01, ***p < 0.001 from two-way ANOVA test (a,b) or one-way ANOVA test (c).