Structures of antitumor organo-IrIII half-sandwich complexes evaluated in vivo previously (IrA-IrE) and in this work (6). TGI = tumor growth inhibition (with respect to the vehicle control). A detailed comparison is provided in Table S4.

X-ray single crystal structure of complexes (a) 4 (CCDC 2252406) and (b) 5 (CCDC 2252407). Ellipsoids are drawn at 50% probability. Hydrogen atoms and counterions have been omitted for clarity. Color codes: C, gray; N, blue; Ir, red; Cl, green.

Representative dose–response curves of complex 5 (a), complex 6 (b), and cisplatin (c) in A2780 and matched cisplatin-resistant A2780 Cis ovarian cancer cells.

Comparison of cellular uptake of complex 6 (a) and cisplatin (b) in cisplatin-sensitive A2780 and cisplatin-resistant A2780 Cis cells.

Comparison of cellular uptake of complex 6 (a) and cisplatin (b) in A549 lung cancer cells and MRC5 lung fibroblasts.

(a) Reaction of complex 4 with GMP and NAC and formation of covalent adducts. (b) Time-dependent NMR spectra of a 1:2 mixture of 4 and GMP incubated at 37 °C in D2O/CD3OD (1/1, v/v). (c) ESI-mass spectrum of a 1:2 mixture of 4 and GMP after 42 h incubation. (d) Time-dependent NMR spectra of a 1:10 mixture of 4 and NAC incubated at 37 °C in D2O/CD3OD (1/1, v/v). (e) ESI-mass spectrum of 1:10 mixture of 4 and NAC after 8 h incubation. Extended stack plots are presented in Figures S28 and S29.

ROS level in HeLa cells either left untreated or treated with complex 6 or H2O2 as a positive control (500 μM, 15 min) measured using the H2DCFDA assay. Data presents the average ± SD of two independent experiments, each performed in six replicates.

Stability of complexes in human plasma.

Complex 6 inhibits blood vessel formation in a dose-dependent manner. (a) Graphic representation of subintestinal venous plexus (SIVP) formation in a wild-type embryo. Red arrows indicate angiogenic sprouts from the posterior cardinal vein (PCV, black arrow), around 28 and 48 hpf (hours post fertilization). Red arrowheads indicate vascular compartments in SIVPs at 72 hpf. (b) Schematic representation of the experimental procedures. (c) Maximum intensity confocal images of the SIVP region at 72 hpf transgenic embryos Tg(etv2:EGFP), expressing EGFP in the endothelial cells of the blood vessels. The SIVP area is shown with yellow dotted lines, and red arrowheads represent the compartments. (d) Dot plot depicting the area covered by SIVP (n = 20 from each treatment condition). (e) Quantification of the number of compartments in SIVPs (n = 20 from each treatment condition). (f) Embryo viability analysis. (g) The bright-field images of the embryos at 3 dpf (dpf = days post fertilization, scale: 1 mm). Figure S32 is the extended image. In panels (d) and (e), data are mean ± standard error of the mean (SEM), and each sample represents one animal.

Complex 6 inhibits VEGFA and BMP signaling-associated gene expression in a dose-dependent manner in zebrafish embryos. (a) Schematic of the experimental procedure. (b) qPCR analysis of vegfaa, vegfab, ccnd1, ccnd2a, ccnd2b, and etv2 in the vehicle and complex 6-treated 72 hpf embryos (n = 4). (c) qPCR analysis of bmp2b, bmp4, bmp7a, and id2a in the vehicle and 6-treated 72 hpf embryos (n = 4). In parts (b) and (c), data are the mean ± SEM. Mean Ct values are presented in Table S3.

Antitumor efficacy of 6 and Lipo-6 in mice bearing the A549 human lung xenograft. (a) Schematic of the A549 xenograft model establishment and treatment schedule. (b) Tumor volume of different treatment groups (n = 5 per group) during the treatment period. (c) Tumor weight of different groups on day 20. (d) Average body weight of mice of different treatment groups. (e) Biodistribution of 6 and Lipo-6, 18 h post last injection showing ∼11-fold higher Ir in tumor and blood for Lipo-6 as compared to complex 6 (see Table S5 in ESI for complete data). Data shown as the mean ± SEM (n = 5 for all groups up to day 20, except the group treated with Lipo-6 where n = 5 up to day 18 and n = 4 on day 20); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Note: the data in 11b, 11c, and 11d for vehicle saline- and cisplatin-treated groups are reproduced with permission from ref 53. Copyright [2023] [John Wiley and Sons]. Because the in vivo study of 6 and a few other anticancer agents including cisplatin were performed together, only one vehicle-treated group and one cisplatin-treated group as a positive control were used for all treatment groups to minimize the number of mice used for ethical reasons.

(a) TEM image of the Lipo-6 nanoparticle. (b) Size distribution of Lipo-6 from DLS measurements. The average hydrodynamic radius is 80 ± 10 nm (average ± SD of two independent measurements). (c) Stability of Lipo-6 at physiological pH 7.4 and acidic pH 6.5 and 5.5.