Ligand Dictated Photosensitization of Iridium(III) Dithiocarbamate Complexes for Photodynamic Therapy.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Ligand Dictated Photosensitization of Iridium(III) Dithiocarbamate\n Complexes for Photodynamic Therapy\n Monika Negi, Tejal Dixit, and V. Venkatesh*\n Cite This: Inorg. Chem. 2023, 62, 20080\u221220095 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: Organelle-targeted photosensitizers (PSs) for photodynamic\n therapy (PDT) are considered as an effective therapeutic strategy for the\n development of next generation PSs with the least side effects and high\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:28:44 (UTC).\n\n\n\n\n therapeutic efficacy. However, multiorganelle targeted PSs eliciting PDT via\n both type I and type II mechanisms are scarce. Herein, a series of\n cyclometalated iridium(III) complexes were formulated [Ir(C\u2227N)2(S\u2227S)]\n (C\u2227N = 2-phenylpyridine (ppy) and 2-(thiophen-2-yl)pyridine (thpy); S\u2227S =\n diethyldithiocarbamate (DEDTC), morpholine-N-dithiocarbamate\n (MORDTC) and methoxycarbonodithioate (MEDTC)) and the newly\n designed complexes Ir2@DEDTC and Ir1@MEDTC were characterized by\n single crystal X-ray crystallography. Complexes containing thpy as C\u2227N ligand\n exhibit excellent photophysical properties such as red-shifted emission, high\n singlet oxygen quantum yield (\u03d5\u0394) and longer photoluminescence lifetime\n when compared with complexes containing ppy ligands. Ir2@DEDTC exhibits the highest \u03d5\u0394 and photoluminescence lifetimes\n among the synthesized complexes. Therefore, Ir2@DEDTC was chosen to evaluate the photosensitizing ability to produce reactive\n oxygen species (ROS). Upon blue light irradiation (456 nm), it efficiently produces ROS, i.e., hydroxy radical (\u2022OH) and singlet\n oxygen (1O2), which was confirmed by electron paramagnetic resonance (EPR) spectroscopy. In vitro photocytotoxicity toward\n HCT116, HeLa, and PC3 cell lines showed that out of all the synthesized complexes, Ir2@DEDTC has the highest photocytotoxic\n index (PI > 400) value. Ir2@DEDTC is efficiently taken up by the HCT116 cell line and accumulated mainly in the lysosome and\n mitochondria of the cells, and after PDT treatment, it elicits cell shrinkage, membrane blebbing, and DNA fragmentation. The\n phototherapeutic efficacy of Ir2@DEDTC has been investigated against 3D spheroids considering its ability to mimic some of the\n basic features of solid tumors. The morphology was drastically altered in the Ir2@DEDTC treated 3D spheroid after the light\n irradiation unleashed the potential of the Ir(III) dithiocarbamate complex as a superior PS for PDT. Hence, mitochondria and\n lysosome targeted photoactive cyclometalated Ir(III) dithiocarbamate complex exerting oxidative stress via both type I and type II\n PDT can be regarded as a dual-organelle targeted two-pronged approach for enhanced PDT.\n\n\n 1. INTRODUCTION irreversible cellular damage.7 Hence, PDT has been extensively\n Chemotherapy is one of the first-line medications for cancer studied and considered as an effective cancer treatment\n treatment.1 An increasing number of reports related to drug strategy.\n resistance and systematic toxicities resulted in the evolution of In recent years, various organic8,9 and inorganic-based PSs\n the field in search of new cancer treatment modalities. Light- have emerged as potent PS that can show excellent\n activated therapy/phototherapy strategy has gained immense photophysical properties, e.g., long fluorescence lifetimes,\n attention in the field of cancer treatment with improved large Stokes shifts, and high photostability.10 In inorganic\n therapeutic potential.2,3 PDT is a light-mediated noninvasive PSs, some organometallic cyclometalated iridium(III) com-\n anticancer strategy that involves a PS and a particular plexes have already been addressed as potent candidates in the\n wavelength of light.4 PDT works by two mechanisms: type I previous literature that has high 1O2 production ability, giving\n and type II.5 In the case of type I PDT, PSs get activated with a them the potency to suppress cancer cells through type II\n specific wavelength of light which is accompanied by electron\n transfer to other biological substrates from the triplet excited\n state of PSs to produce ROS.6 On the contrary, type II PDT Received: August 23, 2023\n involves interactions of the triplet excited state of PS with Revised: November 2, 2023\n surrounding molecular triplet oxygen (3O2) upon light Accepted: November 3, 2023\n irradiation via energy transfer to generate cytotoxic 1O2. Published: November 23, 2023\n These ROS are responsible for oxidative stress in cancer cells,\n which alters the redox homeostasis of cells and leads to\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c02942\n 20080 Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nPDT.11\u221213 However, the clinical applications of many\ncyclometalated iridium(III) complexes are curtailed by their\nlow quantum yield of 1O2 production and short excited state\nlifetime.14 Only a few iridium(III) cyclometalated complexes\nhave been studied, which could show high ROS production by\nundergoing both type I15,16 and type II PDT with long-lived\ntriplet excited states.17,18 Hence, there is a need for the\ndevelopment of PSs, which can be activated by both type I and\nII PDT for effective cancer therapy.\n Additionally, PDT follows localized delivery of PSs and gets\nactivated by specific light irradiation, which can diminish side\neffects on normal cells.19 Because of its spatiotemporal\nspecificity with the least side effects, the development of new\nand effective PSs can improve the therapeutic efficacy in the\nfield of cancer treatment. Phosphorescent iridium(III) cyclo-\nmetalated complexes have been widely reported to be able to\nlocalize at various cellular organelles, such as lysosomes,20\nmitochondria,21\u221223 nuclei,24\u221226 and endoplasmic reticu- Figure 1. Molecular structures of synthesized cyclometalated Ir(III)\nlum.27,28 A few studies have shown that some phosphorescent complexes.\ncyclometalated iridium(III) complexes accumulate in more\nthan one organelle of the cell, such as lysosome and dithiocarbamate (MORDTC) and methoxycarbonodithioate\nmitochondria,29 nucleus and mitochondria,30 etc., and show (MEDTC) was prepared according to previously reported\nbetter therapeutic efficacy. procedures, Scheme S1. Ir(III) dithiocarbamate complexes\n Neutral iridium(III) cyclometalated complexes with various were synthesized by using [(ppy)2-Ir-\u03bc-Cl]2 and [(thpy)2-Ir-\u03bc-\nligands, such as imidoylamidinate,31 \u03b2-diketonate,32 and Cl]2 by following the synthetic route shown in Scheme 1. All\nbenzothiazole,33 were reported as phosphorescence organic\nlight-emitting diodes (OLEDs) as well as liquid droplet (LD)- Scheme 1. Synthetic Route of Ir(III) Dithiocarbamate\nspecific34 phosphorescent probes, respectively. Recently Complexes\ncationic-based Ir(III) cyclometalated complexes have been\nstudied as glutathione sensing agents.35 Also, neutral iridium-\n(III) complexes bearing dithioformic acid ligands were\nreported as mitochondria-targeted cytotoxic agents.36 How-\never, the photophysical properties and their ability to act as PS\nfor PDT have not been explored. Iridium(III) dithiocarbamate\ncomplexes also showed effective carbon disulfide (CS2),37 Hg2+\nsensing,38,39 as well as explored widely in the field of\nOLED.40\u221242 However, these types of complexes are not well\nstudied in terms of their cytotoxic applications.43,44 Herein, we\nhave chosen dithiocarbamate-based ligands for designing\nIr(III) complexes because of the significant role of the sulfur\natom as an efficient triplet harvesting unit.45,46 Five neutral\ncyclometalated iridium(III) complexes, [Ir(ppy)2(DEDTC)]\n(Ir1@DEDTC, DEDTC = diethyldithiocarbamate), [Ir-\n(ppy)2(MORDTC)] (Ir1@MORDTC, MORDTC = morpho-\nline dithiocarbamate), [Ir(ppy)2(MEDTC)] (Ir1@MEDTC,\nMEDTC = methoxycarbonodithioate), [Ir(thpy)2(DEDTC)]\n(Ir2@DEDTC), and [Ir(thpy) 2 (MORDTC)] (Ir2@\nMORDTC), were synthesized. The potential complex Ir2@\nDEDTC was found to localize in mitochondria and lysosomes complexes are incorporated with one S\u2227S ligand and two C\u2227N\nand exerts PDT via both type I and type II mechanisms. The ligands. We anticipate that incorporating sulfur in the form of\nstructures of synthesized cyclometalated Ir(III) complexes are [(thpy)2-Ir-\u03bc-Cl]2 dimer will enhance its photophysical\nlisted in Figure 1. The photophysical properties, cellular properties, higher triplet excited state lifetime, photocytotox-\nuptake, 1O2 production, photocytotoxicity, and bioimaging icity, and cellular imaging ability of the complexes.47 All the\napplications were evaluated. synthesized complexes were further characterized by 1H NMR,\n 13\n C NMR, and electrospray ionization (ESI-MS) spectroscopy\n2. RESULTS AND DISCUSSION (Figures S1\u2212S21), as well as single crystal XRD.\n 2.1. Synthesis and Characterization. In this work, the 2.1.1. X-ray Characterization. The single crystals of Ir1@\ninvestigated dithiocarbamate-based cyclometalated Ir(III) MEDTC and Ir2@DEDTC suitable for X-ray diffraction\ncomplexes were synthesized in two steps. First, The \u03bc- (XRD) were crystallized by slow evaporation method with\nchloride-bridged dimer Ir(III) complexes [(ppy)2-Ir-\u03bc-Cl]2 DCM-toluene (1:1, v/v) and ether diffusion method,\nand [(thpy)2-Ir-\u03bc-Cl]2 were synthesized from iridium(III) respectively. The crystal structures of both Ir(III) complexes\nchloride (precursor) using previously reported literature with are shown in Figure 2. The complexes Ir1@MEDTC and Ir2@\nminor modifications. Also, the potassium salt of morpholine DEDTC were crystallized in monoclinic space groups P21/n\n 20081 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n most of the reported cyclometalated iridium(III) com-\n plexes.52,53\n The fluorescence emission spectra for synthesized complexes\n were recorded in CH3CN and DMSO-water (0.4%, v/v) at\n 298 K, are shown in Figure 3(b) and Figure S22(b). The\n emission maximum wavelengths of the synthesized Ir(III)\n complexes were almost the same in these solvents. The PL\n spectra of complexes Ir1@DEDTC, Ir1@MORDTC, and\n Ir1@MEDTC showed a broad green phosphorescent emission\n with maxima at 550 nm upon excitation at 405 nm. Moreover,\n complexes Ir2@DEDTC and Ir2@MORDTC showed an\nFigure 2. X-ray structures of complexes Ir1@MEDTC and Ir2@ orange-red phosphorescent emission at 610 nm with a less\nDEDTC (left to right).\n intense narrow band at around 655 nm upon excitation at 450\n nm. It is worth noting that these two complexes, i.e., Ir2@\nand P21/c, respectively. The selected bond lengths in each DEDTC and Ir2@MORDTC, exhibited a red-shifted (bath-\ncomplex are represented in Table S1. The details of single ochromic) emission maxima of 100 nm compared to Ir1@\ncrystal data refinement parameters are summarized in Table DEDTC, Ir1@MORDTC, and Ir1@MEDTC complexes,\nS2. suggesting the crucial role of different C\u2227N ligands in\n 2.2. Photophysical Properties. The UV\u2212visible absorp- modulating the photophysical properties. All of the complexes\ntion spectra of all of the complexes in dry acetonitrile showed a minor solvatochromic effect and a large Stokes shift\n(CH3CN) and DMSO\u2212water (0.4%, v/v) at 298 K are of about 150 nm. It has been extensively studied that a mixture\nshown in Figure 3(a) and Figure S22(a), respectively. The of 3MLCT and ligand-centered 3(\u03c0\u2212\u03c0*) excited states is\ncomplexes display a strong absorption band in the region of responsible for phosphorescence in iridium(III) cyclometa-\n250\u2212300 nm, which is assigned to spin-allowed ligand- lated complexes.54 Moreover, all the synthesized complexes\ncentered or interligand (1\u03c0\u2212\u03c0*) transitions.48,49 In addition, also showed excellent photostability as there was no visible\nthe weak and broad absorption bands (less intense) in the change in the absorption peak under light irradiation (456 nm,\nregion of 350\u2212500 nm are designated to the mixed singlet and 10 mW/cm2) as well as under dark conditions for 48 h Figures\ntriplet metal-to-ligand charge transfer (MLCT) or d\u03c0(Ir)\u2212\u03c0*- S23 and S24. Excellent photostability is one of the important\n(L) (spin allowed and spin forbidden).50,51 The absorption factors for a PS to generate ROS for hampering cancer cells.\nspectra for the synthesized Ir(III) complexes are analogous to The photophysical properties of all of the synthesized\n\n\n\n\nFigure 3. Absorption spectra (a) and emission spectra (b) of Ir(III) complexes (20 \u03bcM) recorded in CH3CN at room temperature. (c)\nFluorescence lifetime spectra of Ir2@DEDTC (Excitation pulse: 450 nm).\n\n 20082 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\niridium(III) complexes are illustrated in Table 1. The excellent ligand. The sulfur-containing C\u2227N ligands along with electron-\nphotostability under light and dark conditions and longer rich S\u2227S ancillary ligand facilitate better intersystem crossing\nabsorption wavelength of complexes suggested that they could due to smaller singlet\u2212triplet energy gap. The enhanced triplet\nbe used as effective PSs for PDT. sensitization significantly influences the phosphorescence\n wavelength, excited state dynamics, and photosensitization\nTable 1. Photophysical Properties of Ir(III) abilities of Ir2@DEDTC and Ir2@MORDTC. It also exhibits a\nDithiocarbamate Based Complexes remarkably higher 1O2 generation quantum yield, which\n indicates the phosphorescent emission nature of the\n complex \u03bbexc/nm \u03bbem/nm \u03d5aem \u03d5(1O2)b \u03c4c/ns complexes.\n Ir1@DEDTC 405 550 0.107 0.281 1.3 2.3. 1O2 Production and Quantification. As iridium(III)\n Ir1@MORDTC 405 550 0.0046 0.239 2.18 complexes are prone to show type II photosensitization by\n Ir1@MEDTC 365 550 0.085 0.271 5.78 quenching the triplet excited state with molecular oxygen to\n Ir2@DEDTC 450 610 0.025 0.864 146 generate 1O2, the ability of synthesized Ir(III) dithiocarbamate\n Ir2@MORDTC 450 610 0.022 0.705 109 complexes to generate 1O2 was studied with the help of 1,3-\na\n Emission quantum yield considering [Ru(bpy)3]Cl2 as reference diphenylisobenzofuran (DPBF) as a 1O2 scavenger in DMSO.\ncompound in deareated CH3CN. b1O2 quantum yield in DMSO (MB Upon blue light irradiation, Ir(III) complexes produce 1O2,\nas reference). cAverage photoluminescence lifetime in aerated DMSO which readily oxidizes DPBF to 1,2-dibenzoyl benzene (DBB).\nas solvent.\n It resulted in the decrement of the DPBF absorbance peak\n maxima at 417 nm, which was used to quantify the production\n 2.2.1. Lifetime Measurement. Most of the transition metal of 1O2. The absorbance changes of DPBF alone with\ncomplexes comprise an intermediate lifetime of hundreds of ns irradiation (456 nm) were recorded to show the degradation\nto several \u03bcs because of mixed singlet\u2212triplet states.55 The of DPBF without the complexes and reference compound. The\naverage fluorescence lifetime of most of the Ir(III) complexes absorbance of DPBF declined weakly after irradiation for 40 s\nis a key factor in the production or generation of ROS, such as due to the photobleaching of DPBF alone (Figure S26).\n1\n O2.56 For Type II PDT, a longer triplet excited-state lifetime is Methylene blue (MB) is used as a reference PS while\nneeded to transfer energy to the molecular oxygen. The performing this assay. The absorbance maxima of DPBF\ninclusion of higher molecular weight atoms, such as sulfur, is decreases after the addition of PS with an increase in\nattributed to spin\u2212orbit coupling, which eventually leads to a irradiation time.60 Figure 4(a) shows a steady decrease in\nlonger triplet state lifetime.57\u221259 Ir2@DEDTC showed a the absorption maxima of DPBF in the presence of Ir2@\nsignificantly longer fluorescence lifetime (146 ns) than Ir1@ DEDTC under light irradiation after every 5 s. The values of\nDEDTC (1.3 ns) Figure 3(c) and Figure S25 in aerated \u03d5\u0394 for complexes were estimated by plotting the scatter plot\nDMSO due to the incorporation of the sulfur atom in the C\u2227N for change in absorbance of DPBF for all complexes and\n\n\n\n\nFigure 4. (a) Absorption changes of DPBF with Ir2@DEDTC after light irradiation (0\u221240 s). (b) Photooxidation of DPBF by complexes and the\nreference compound under light irradiation (450 nm, 10 mW/cm2). (c) Ir2@DEDTC with TEMP in dark (gray line) and after 450 nm light\nirradiation for 20 min (red line). (d) Ir2@DEDTC with DMPO in dark (gray line) and after 450 nm light irradiation for 20 min (red line).\n\n 20083 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 2. Photo-Cytotoxicities of Metal Complexes (\u03bcM) in Comparison to Cisplatin against HCT116 (Human Colorectal\nCarcinoma), HeLa (Human Cervical Carcinoma), and PC3 (Human Prostate Carcinoma) Cell Lines Expressed as Mean \u00b1\nStandard Error (n = 3)\n HCT116 HeLa PC3\n a b a b\n compound dark light PI dark light PI dark lighta PIb\n Ir1@DEDTC 49.86 \u00b1 3.10 1.75 \u00b1 1.73 28 86.88 \u00b1 4.2 22.73 \u00b1 3.5 3.8 >100 >100 n.a.\n Ir1@MORDTC 85.44 \u00b1 3.28 4.51 \u00b1 2.60 18.8 >100 50.88 \u00b1 2.2 1.96 50.9 \u00b1 6.2 45.3 \u00b1 5.7 1.13\n Ir1@MEDTC 39.88 \u00b1 6.0 27.09 \u00b1 7.4 1.5 >200 33.24 \u00b1 1.8 6.02 90.04 \u00b1 3.9 39.68 \u00b1 5.2 2.3\n Ir2@DEDTC 44.03 \u00b1 5.83 0.1095 \u00b1 1.2 >400 >300 4.12 \u00b1 1.9 73 >100 58.91 \u00b1 3.7 1.7\n Ir2@MORDTC 25.98 \u00b1 7.3 0.91 \u00b1 2.3 28.6 >100 4.49 \u00b1 2.7 22.3 >100 63.5 \u00b1 4.7 1.59\n Cis-Pt 20.77 \u00b1 5.2 19.66 \u00b1 6.3 1.05 17.64 \u00b1 0.4.6 16.61 \u00b1 4.1 1.06 10.5 \u00b1 3.7 10.27 \u00b1 2.9 1.02\na\n Irradiated at 456 nm LED light (30 mW/cm2) for 40 min. bPhotocytotoxicity index (PI = IC50(dark)/ IC50(light)) indicates the efficiency of the\nlight treatment.\n\n\n\n\nFigure 5. Cell viability was studied in HCT116(a), HeLa (b), and PC3 (c) cancer cells formulated with different concentrations of Ir2@DEDTC\nunder dark and light conditions. Values are represented as the mean \u00b1 SD of three independent experiments.\n\nreference compounds for different irradiation periods Figure generated by the complex Ir2@DEDTC. Upon irradiation with\n4(b). The results showed that the trend of the quantum yield 450 nm light for 20 min, the deaerated CH3CN solutions of\nfor the generation of 1O2 is as follows: Ir2@DEDTC (0.864) > Ir2@DEDTC (100 \u03bcM) and TEMP (50 mM), a three-line\nIr2@MORDTC (0.705) > Ir1@DEDTC (0.281) > Ir1@ signal with equal intensities (1:1:1) emerged between 3300\u2212\nMEDTC (0.271) > Ir1@MORDTC (0.239). Herein, Ir2@ 3400 G magnetic field Figure 4(c). A hyperfine splitting\nDEDTC and Ir2@MORDTC showed significantly higher 1O2 constant of A = 15 G and a g factor of 2.0141 are observed for\nquantum yields than others, indicating that they could be the TEMP/1O2 adduct. To verify the ability to generate other\nefficient PSs. The UV\u2212vis spectra of change in absorbance of ROS by the Ir(III) complexes after light irradiation, we\nDPBF with complexes with the thpy ligand showed a higher employed DMPO as the trapping agent for O2\u2022\u2212 and \u2022OH.\ndecline as compared to complexes containing ppy, suggesting a Figure 4(d) represents a four-line spectrum, which is the\nremarkable and rapid generation of ROS (Figure S26). characteristic spectrum of the DMPO/\u2022OH adduct. Also, the\n The ROSs generated by the triplet excited state PSs via the generation of \u2022OH is further confirmed by the BMPO/\u2022OH\ntype I (electron transfer) or type II (energy transfer) pathway adduct Figure S27. These results indicated that Ir2@DEDTC\nof PDT cannot be identified by EPR directly because of their is capable of generating 1O2 and \u2022OH after irradiation with 450\nshort lifetimes. EPR is a sensitive and reliable technique to nm light. The triplet excited state of a PS yields ROS only after\ndetect and identify the type of ROS with the help of spin- light irradiation. No signals in EPR were observed in the dark\ntrapping agents such as 2,2,6,6-tetramethylpiperidine (TEMP) (Ir2@DEDTC without light irradiation). The results indicated\nfor 1O2, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for other that synthesized complexes generate ROS after irradiation with\nROS species (O2\u2022\u2212 and \u2022OH) and 5-tert-butoxycarbonyl-5- 450 nm and could act as type I and type II PS.\nmethyl-1-pyrroline-N-oxide (BMPO) for \u2022OH and thiyl (RS\u2022) 2.4. In Vitro Photocytotoxicity and Cellular Uptake.\nradical. The stable spin adducts, which are readily formed by After confirming that Ir(III) complexes undergo type I and\nreacting ROS with these diamagnetic spin traps, can be type II photosensitization after light irradiation, we set out to\ncharacterized and identified by EPR analysis. Herein, the EPR investigate whether it could show photocytotoxicity against\nspin trapping technique is utilized to confirm further the ROSs different cell lines by using cell viability assay (MTT assay).\n 20084 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 6. (a\u2212e) Bright-field images of HCT116 captured after DCFH-DA staining. Cells alone without light, cells alone with light (456 nm, 30\nmW/cm2, 20 min), hydrogen peroxide (50 \u03bcM, positive control), Ir2@DEDTC (20 \u03bcM, without light), and Ir2@DEDTC (20 \u03bcM, with light) (left\nto right). (f\u2212j) Corresponding fluorescence was obtained under the green channel. Magnification: 40\u00d7. Scale bar: 20 \u03bcm. (k) Quantitative\nstatistical analysis of the production of ROS in the cells. Data expressed as mean \u00b1 SD (n = 2), p values were measured using one-way ANOVA\nwith Turkey multiple comparison test, ***p < 0.001.\n\nThe IC50 (half-maximum inhibitory concentration) values with plasma mass spectrometry (ICP-MS). The amount of Ir(III)\nphototoxicity index (PI) values of synthesized complexes accumulated inside the cells (Ir content per 105 cells) is\ntoward various cell lines are summarized in Table 2. Low dark calculated with the help of standard graphs Figure S29.\ntoxicity was observed for HCT116, HeLa, and PC3 cells after HCT116 cells were incubated with 20 \u03bcM Ir2@DEDTC with\n24 h of drug exposure and 24 h recovery. All the complexes different incubation time intervals, i.e., 2, 4, 6, and 12 h. The\nwere observed to be less toxic under dark conditions (IC50 = result showed that intracellular Ir(III) content increases with\n44.03\u221285.44 \u03bcM). Upon light exposure, Ir2@DEDTC was increasing incubation time intervals, i.e., 2 h (131.6 nM Ir/105\nfound to be highly cytotoxic (IC50 = 0.1095 \u03bcM, PI > 400) cells) < 4 h (247.55 nM) < 6 h (249.9 nM) < 12 h (326.2\nagainst the HCT116 cell line by generating ROS. Complexes nM). To understand the selectivity of Ir2@DEDTC for\nwith thpy ligand, i.e., Ir2@DEDTC and Ir2@MORDTC, HCT116 over PC3 cells, the uptake experiment was performed\ndisplayed higher cytotoxicity (lower IC50) than cisplatin after with PC3 cells. After 6 h of treatment, only 18.4 nM Ir/105\nlight irradiation for 40 min against HCT116, HeLa cell lines as cells accumulated in PC3 cells, when compared with 249.9 nM\ncompared to PC3 cancer cell line. Additionally, Complexes Ir/105 cells accumulated in HCT116 cells (Table S5 and\nalso showed lower dark toxicity toward normal WRL-68 cell Figure S30). Hence, the lower uptake of complexes in the PC3\nlines than dithiocarbamate (DTC) ligand alone Tables S3 and cell line could be the reason for the least dark toxicity and\nS4. Overall, our studies reflect the potential of Ir2@DEDTC photocytotoxicity of the complexes.\nand Ir2@MORDTC to act as potential PSs for PDT after 2.5. Intracellular ROS Production (DCFH-DA Assay).\nirradiation with blue LED (456, 30 mW/cm2 light) against ROS is an important factor in estimating PS-induced\nHCT116 and HeLa cancer cell lines Figures 5 and S28. In cytotoxicity after light irradiation. To detect the generation\ncontrast to the cytotoxicity studies of iridium(III) dithiocarba- of ROS inside cells, a cellular ROS indicator, i.e., 2,7-\nmate complexes reported previously,36 in our experiment, we dichlorodihydrofluorescein diacetate (DCFH-DA), is used to\nhave noted that on the treatment of complexes for 24 h, evaluate the ROS generation ability of Ir2@DEDTC in the\nfollowed by 24 h recovery, all the complexes exhibited poor HCT116 cell line in dark and light conditions with all the\ndark toxicities (due to proliferation of cells during recovery necessary control experiments. After cellular internalization,\nperiod). It is also evident that the light induced cytotoxicities nonfluorescent DCFH-DA gets hydrolyzed to DCFH and\nof the complexes are significant even after the recovery period. further oxidized into a green fluorescent compound, i.e.,\nAs Ir2@DEDTC has shown the maximum photocytotoxicity dichlorofluorescein (DCF), in the presence of ROS.61\nefficiency, it has been used as a lead molecule to carry out HCT116 cells treated with Ir2@DEDTC, followed by 456\nfurther investigations. nm light irradiation, showed bright green fluorescence\n As the synthesized Ir(III) dithiocarbamate complexes emission from cells Figure 6(j), which is better than that of\nshowed better photocytotoxicities against HCT116 cell line, the positive control (H2O2 treated cells) Figure 6(h), whereas,\ntherefore the cellular uptake studies for Ir2@DEDTC were under dark conditions, green emission was observed from very\ninvestigated in the same cell line using inductively coupled few cells, Figure 6(i). Similarly, untreated cells with and\n 20085 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 7. Nuclear fragmentation and chromatin condensation studies by DAPI staining. (a\u2212e) Bright-field images obtained after DAPI staining for\ncells alone without light, cells alone with light (456 nm, 30 mW/cm2, 20 min), Cisplatin (20 \u03bcM, positive control), Ir2@DEDTC (20 \u03bcM, without\nlight), and Ir2@DEDTC (20 \u03bcM, with light) (left to right). (f\u2212j) Corresponding fluorescence was obtained under the blue channel. Magnification:\n40\u00d7. Scale bar: 20 \u03bcm. Arrows represent chromatin condensation and nucleus disintegration.\n\nwithout light irradiation exhibit very weak fluorescence\nemission, Figure 6(f),(g). The fluorescence intensity of DCF\n(\u03bbex = 485 nm, \u03bbem = 535 nm) is correlated with the fold\nincrease in ROS production, which is measured by quantifying\nthe fluorescence emission with the help of a microplate reader.\nUpon 20 min of light irradiation at 456 nm, the fluorescence\nintensity is increased about 6-fold in the presence of Ir2@\nDEDTC, indicating a large amount of ROS production in the\ncells as compared to untreated cells (Figure 6(k)). Therefore,\nthese results signify that PDT-mediated ROS production is\ninvolved in apoptotic cell death caused by cyclometalated\nIr(III) complex.\n 2.6. DNA Fragmentation Studies. Nuclear fragmentation\nstudy was performed by using 4\u2032,6-diamidino-2-phenylindole\n(DAPI) to investigate the cause of cell death after light\n Figure 8. Morphology changes of HCT116 upon incubation with\nirradiation (456 nm, 30 mW/cm2, 20 min). The cells in the Ir2@DEDTC under light and dark conditions. Cells alone (a) without\ncontrol group (untreated cells with and without light) and light and (b) with light (456 nm, 30 mW/cm2, 20 min) and Ir2@\nIr2@DEDTC without the light group showed no sign of DNA DEDTC (20 \u03bcM) (c) with and (d) without light. Magnification: 40\u00d7.\ndisintegration (chromatin condensation) Figure 7(f),(g),(i). Scale bar: 20 \u03bcm. (Arrows represent cell shrinkage, membrane blebs,\nCisplatin (Cis-Pt) is used as a positive control, which shows and rounding of cells).\nsome chromatin condensation with high fluorescence intensity\nfor DAPI (Figure 7(h)). The morphological changes of the trates inside the viable cells easily and gets hydrolyzed into\nnucleus in the case of cells incubated with Ir2@DEDTC (20 green emissive fluorescein by esterases present in the\n\u03bcM) in the presence of light showed condensed chromatin and cytoplasm of live cells,62 whereas PI is nonpermeable through\nhigher blue fluorescence intensity (Figure 7(j)). The presence the cell membrane and can intercalate with DNA for\nof a greater extent of nuclear fragmentation in the Ir2@ membrane-compromised cells (dead cells) and give red\nDEDTC+Light group, as compared to other groups, indicated fluorescence after binding to DNA. In Figure 9(f),(g), most\nthat the cells were undergoing apoptosis through DNA of the cells are viable (showing green fluorescence) in the case\ndamage. of untreated cells with and without light irradiation. Cells\n 2.7. Cell Morphology Change and Live/Dead Cell incubated with Ir2@DEDTC under dark conditions showed\nStaining. The apoptotic morphological changes, such as few dead cells and more live cells (Figure 9(r)). Upon\nmembrane blebbing, cellular fragmentation, and cell shrinkage, irradiation with 456 nm light, the cells showed high red\nwere investigated. The morphology of HCT116 cells in the fluorescence of PI after 24 h as compared to 6 h of incubation\ncontrol group (without Ir2@DEDTC treatment) showed with Ir2@DEDTC Figure 9(s),(t).\ninsignificant changes with and without light conditions (Figure 2.8. Cellular Uptake and Intracellular Localization\n8(a),(b)). No changes in morphology were observed in the Studies. The cellular uptake of Ir2@DEDTC in HCT116\ncase of HCT116 cells treated with Ir2@DEDTC under dark cells was visualized using fluorescence microscopy after 8 h of\nconditions (Figure 8(c)). On the other hand, most of the cell incubation (Figure S31). The intracellular colocalization of\nmorphology has been dramatically changed after treatment Ir2@DEDTC was investigated by colocalization assay in the\nwith Ir2@DEDTC under light irradiation (456 nm, 30 mW/ presence of Mitotracker Green (Mitochondrial staining dye,\ncm2, 20 min, Figure 8(d)). This suggests that Ir2@DEDTC MTG), Lysotracker Green (Lysosomal staining dye, LTG),\ncan efficiently induce cellular apoptosis under light irradiation. and DAPI (nuclear stain) against HCT116 cell line. The\n To further verify the photocytotoxic profile of Ir2@DEDTC, merged images in Figure 10(d),(h) show the selective\nfluorescein diacetate (FDA) and propidium iodide (PI) colocalization of MTG (Pearson\u2019s R value = 0.76) and LTG\ncostaining assay was performed. Nonfluorescent FDA pene- (R = 0.81) with complex Ir2@DEDTC. In contrast, costain\n 20086 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 9. Live/dead costained HCT116 cells treated with Ir2@DEDTC for 6 and 24 h with or without light irradiation (456 nm, 30 mW cm\u22122, 20\nmin). (a\u2212e) Bright-field images of HCT116 captured after FDA/PI staining. Cells alone without light, cells alone with light, Ir2@DEDTC (20 \u03bcM,\nwithout light after 24 h), Ir2@DEDTC (20 \u03bcM, light irradiated after 6 h of incubation), and Ir2@DEDTC (20 \u03bcM, light irradiation after 24 h of\nincubation) (left to right). (f\u2212j) Corresponding fluorescence was obtained under the green channel. (k\u2212o) Corresponding fluorescence of PI was\nobtained under the red channel. (p\u2212t) Merge images of green and red channels of the respective groups (green fluorescence: viable cells; red\nfluorescence: dead cells). Magnification: 40\u00d7. Scale bar: 20 \u03bcm.\n\n\n\n\nFigure 10. Colocalization assay with HCT116 cells incubated with Ir2@DEDTC costained with specific organelle targeting dye analyzed by\nfluorescence microscopy. (a\u2212d) Bright-field, green channel (LTG), red channel (Ir2@DEDTC, 20 \u03bcM), and merged images of the cells incubated\nwith Ir2@DEDTC (20 \u03bcM) and stained with LysoTracker Green (LTG, 500 nM) (left to right). scale bar: 10 \u03bcm. (e\u2212h) Bright-field, green\nchannel (MTG), red channel (Ir2@DEDTC, 20 \u03bcM), and merged images of the cells incubated with Ir2@DEDTC (20 \u03bcM) and stained with\nMitoTracker Green (MTG, 500 nM) (left to right). (i\u2212l) Bright-field, blue channel, and red channel (Ir2@DEDTC, 20 \u03bcM) and stained with\nDAPI (1 \u03bcg/mL) (left to right). Magnification: 60\u00d7 (DIC). Scale bar: 5 \u03bcm.\n\n Hence, we confirmed that Ir2@DEDTC was taken up\npatterns of Ir2@DEDTC matched poorly with DAPI (Figure effectively by the cells and predominantly localized in the\n10(l)). lysosome and mitochondria of the cells. Also, the photo-\n 20087 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 11. JC-1 stained HCT116 cells treated with Ir2@DEDTC for 6 h with or without light irradiation. (a\u2212d) Bright-field images of HCT116\ncaptured after JC-1 staining. Cells alone without light (a), cells alone with light (b), Ir2@DEDTC (20 \u03bcM, without light) (c), and Ir2@DEDTC\n(20 \u03bcM, with light) (d) (up to down). (e\u2212h) Corresponding fluorescence of JC-1 monomer was obtained under the green channel. (i\u2212l)\nFluorescence of JC-1 aggregate was obtained under the red channel. (m\u2212p) Merge images of green and red channels of the respective groups\n(green fluorescence: low MMP, red fluorescence: high MMP). Magnification: 40\u00d7. Scale bar: 20 \u03bcm.\n\ncytotoxic behavior of the synthesized Ir(III) dithiocarbamate phases (G0/G1, S, Sub G1, and G2/M) for untreated cells (a),\ncomplexes may originate from lysosome and mitochondria- Cisplatin as a positive control (b), and Ir2@DEDTC in dark\ntargeted cell death. (c) and light conditions (d) after 24 h. Figure 12(e) represents\n 2.9. Induction of Cellular Apoptosis and Loss of the bar graph representation of the distribution of the cells in\nMitochondrial Membrane Potential (MMP). Based on the all the phases. Cells in the Sub G1 phase correspond to\ncolocalization images by a fluorescence microscope, we further apoptotic cells having fragmented DNA, which emits less\nevaluated the impact of complex Ir2@DEDTC on mitochon- fluorescence of PI. A prominent increase in the population of\ndrial membrane integrity in dark and light conditions against cells in Sub G1 phase is observed in the case of cells treated\nHCT116. Mitochondria dysfunction is an important hallmark with Ir2@DEDTC for 24 h and then irradiated for 40 min. On\nof an intrinsic apoptotic pathway of the cell. 5,5\u2032,6,6\u2032- the other hand, untreated cells and Ir2@DEDTC under dark\nTetrachloro-1,1\u2032,3,3\u2032-tetraethylimidacarbocyanine iodide (JC- conditions showed a lesser fraction of cells in Sub G1 phase,\n1) is a fluorescent dye that forms J-aggregates and emits red revealing that cells are undergoing an apoptotic phase after\nfluorescence for intact and healthy mitochondria (high MMP). light irradiation. The fraction of cells in the G0/G1 phase is\nIn contrast, JC-1 dye exists as a monomer and shows green higher for Ir2@DEDTC+Light group as compared to other\nfluorescence for mitochondria having a depolarized membrane phases, and hence it reveals that the cells are undergoing cell\n(low MMP). In Figure 11, red fluorescence is observed for cycle arrest at G0/G1 phase. On the other hand, Cisplatin\nuntreated cells under light and dark conditions. Also, Ir2@ treated cells (20 \u03bcM) showed cell cycle arrest in the G2M\nDEDTC, under dark conditions, showed red fluorescence, phase (Figure 12(e)). Interestingly, the high population of cells\nwhich depicts the mitochondrial integrity of the cells. In in G0/G1 for complex Ir2@DEDTC under light (30 mW/cm2,\ncontrast, Ir2@DEDTC with light (456 nm, 30 mW/cm2, 20 40 min) supports G0/G1 cell cycle arrest responsible for the\nmin) irradiation extensively showed green fluorescence high cytotoxicity of the synthesized Ir(III) complexes.\ncorresponding to the JC-1 monomer. Hence, complex Ir2@ 2.10. 3D Multicellular Tumor Spheroids Imaging.\nDEDTC-treated cells are undergoing mitochondrion-mediated Based on the results obtained from photocytotoxicity assays in\napoptosis by lowering the mitochondrial membrane potential. 2D monolayer cells, the potency of Ir2@DEDTC was\n The lowering of mitochondria membrane potential further evaluated against 3D multicellular tumor spheroids (MCTS)\nsuggested us to investigate the cell cycle disturbance by in the presence of light irradiation. To mimic the solid tumor\ncomplex Ir2@DEDTC under dark and light conditions. The environment, MCTS has appeared to be an in vitro model for\nimpact of Ir2@DEDTC on cell cycle distribution is evaluated evaluating the therapeutic potential of the drug. Like most\nwith the help of a flow cytometer with propidium iodide (PI) solid tumors, MCTS shows a heterogeneous surrounding with\nstaining. Figure 12 shows the distribution of cells in various a necrotic core at the center having hypoxic regions (oxygen\n 20088 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 12. Flow cytometry analysis with PI staining in HCT116 cells treated with Ir2@DEDTC for 24 h with or without light irradiation. Cells\nalone (a), Cisplatin (b), Ir2@DEDTC (20 \u03bcM, without light) (c), and Ir2@DEDTC (20 \u03bcM, with light) (d). (e) Corresponding bar graph\nrepresents the cell population in G0/G1, Sub G1, S, and G2/M phases for all of the respective groups.\n\ndeficient).63 Because of these factors, most of the drugs show presence of light, Ir2@DEDTC exhibits potent photocytotox-\ngreat cytotoxicity on 2D monolayer cells but undergo drug icity in the MCTS model and induces cellular apoptosis.\nresistance while performing in vivo studies. Hence, MCTS\nstudies are the best model to show the therapeutic efficacy of\nthe drug. The loss in the integrity of the spheroids was\n \u25a0 CONCLUSIONS\n In summary, we have synthesized a series of cyclometalated\nobserved in Ir2@DEDTC after light irradiation when iridium(III) dithiocarbamate complexes with different C\u2227N\ncompared with the untreated spheroids as well as spheroid and S\u2227S ligands. Out of the five synthesized complexes, two\ntreated with Ir2@DEDTC under dark conditions Figure S32. complexes, Ir2@DEDTC and Ir2@MORDTC, exhibited\nThese results indicated that a combination of Ir2@DEDTC excellent photophysical properties such as red-shifted\nand light showed cytotoxicity against MCTS. fluorescence emission, higher luminescence lifetime, and 1O2\n To further investigate cell death in MCTS, a live/dead quantum yield. The role of ligands in dictating the photo-\ncostaining assay was performed with the help of FDA and PI sensitizing ability of the complexes was evident from their\ndye. The fluorescence images were captured in the green (for photocytotoxicity assay. Ir2@DEDTC exhibits the highest\nFDA) and red channels (for PI) after 24 h incubation with photocytotoxic index against the HCT116 cell line and is\nIr2@DEDTC followed by light treatment. The Ir2@DEDTC found to be least toxic to normal human hepatic cells (WRL-\n+Light group showed fewer cells stained in the green channel 68). Also, Ir2@DEDTC was efficiently taken up by HCT116\nwhen compared with other control groups. Also, the number of cells and predominantly localized in the lysosome and\ncells stained with PI in red channel was significantly higher in mitochondria. Furthermore, a combination of Ir2@DEDTC\nthe Ir2@DEDTC+Light group as compared to untreated and light-induced apoptosis through lowering of the\nMCTS (CA and CA+Light) as well as Ir2@DEDTC under mitochondrial membrane potential and further induction of\ndark conditions (Figure 13). These results depicted that in the apoptosis was studied with JC-1 and cell cycle arrest analysis,\n 20089 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n purchased from the National Centre for Cell Science (NCCS),\n Pune, Maharashtra, India. Dulbecco\u2019s modified Eagle\u2019s medium\n (DMEM) along with 10% fetal bovine serum (FBS), 2.2 g/L sodium\n bicarbonate, streptomycin (100 mg/mL) and penicillin G (100 units/\n mL) is used to grow the cells. Fetal bovine serum (FBS) was\n purchased from Gibco (Thermo Fisher Scientific). All the cell lines\n were cultured at approximately 70\u221280% confluence and kept in a\n humidified incubator at 37 \u00b0C with 5% CO2 in cell culture flasks with\n different sizes or plates (tissue cultured), depending on the type of\n experiments to be performed.\n 4.2. Synthesis of Ir(III) Complexes. 4.2.1. Synthesis of [(ppy)2-\n Ir-\u03bc-Cl]2 Dimer. Iridium dimer [(ppy)2-Ir-\u03bc-Cl]2 is synthesized by\n previously reported literature with minor modifications.64 Briefly,\n iridium(III) chloride (100 mg, 0.275 mmol, 1 equiv) hydrate was\n suspended in 2-ethoxyethanol/water (3:1), and 2-phenylpyridine\n (107 mg, 0.68 mmol, 2.5 equiv) was added. The reaction mixture was\n refluxed overnight to obtain a yellow precipitate. The formed\n precipitate was filtered and then washed several times with ethanol\n and further with diethyl ether to get a bright yellow solid (yield 61%).\n 1\n H NMR (500 MHz, DMSO-d6) \u03b4 9.81 (d, J = 5.1 Hz, 2H), 9.53 (d, J\n = 4.9 Hz, 2H), 8.26 (d, J = 8.0 Hz, 2H), 8.18 (d, J = 7.7 Hz, 2H), 8.10\n (t, J = 7.0 Hz, 2H), 8.01 (t, J = 7.0 Hz, 2H), 7.79 (d, J = 6.7 Hz, 2H),\n 7.73 (d, J = 6.9 Hz, 2H), 7.57 (t, J = 6.0 Hz, 2H), 7.45 (t, J = 5.9 Hz,\n 2H), 6.90 (t, J = 6.9 Hz, 2H), 6.84 (t, J = 6.9 Hz, 2H), 6.77 (t, J = 6.8\n Hz, 2H), 6.69 (t, J = 6.8 Hz, 2H), 6.25 (d, J = 6.8 Hz, 2H), 5.66 (d, J\n = 6.9 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) \u03b4 167.36, 167.00,\nFigure 13. Live/dead costained HCT116 MCTS treated with Ir2@ 152.14, 152.09, 150.68, 145.41, 143.80, 143.22, 139.26, 138.20,\nDEDTC, with and without light irradiation (456 nm, 30 mW cm\u22122, 131.29, 129.97, 129.69, 129.01, 124.86, 123.86, 123.62, 122.89,\n20 min). (a\u2212d) Bright-field images of spheroids captured after FDA/ 122.31, 122.02, 120.13, 119.50.\nPI staining. Cells alone (represented as CA) without light, cells alone 4.2.2. Synthesis of MORDTC. The potassium salt of morpholine\nwith light, Ir2@DEDTC (20 \u03bcM, dark), and Ir2@DEDTC (20 \u03bcM, dithiocarbamate was prepared by already reported literature with\nlight irradiated after 24 h incubation) (left to right). (e\u2212h) minor modifications.65 Morpholine (500 mg, 5.7 mmol, 1 equiv) and\nCorresponding fluorescence was obtained under the green channel. KOH (322 mg, 5.7 mmol, 1 equiv) were suspended in 5 mL of\n(i\u2212l) Corresponding fluorescence of PI was obtained under the red ethanol, and the reaction mixture temperature was maintained at 0\nchannel. (m\u2212p) Merge images of green and red channels of the \u00b0C. After that, 200 \u03bcL of carbon disulfide (CS2) was added until the\nrespective groups. Magnification: 10\u00d7. Scale bar: 100 \u03bcm. solution color changed to light yellow and stirred vigorously for 1 h to\n get a white precipitate. Then it was washed with cold diethyl ether\nrespectively. Finally, the photosensitization ability of Ir2@ and dried under a vacuum to get the product as a white solid. (Yield\n 86%). 1H NMR (500 MHz, DMSO-d6) \u03b4 4.33\u22124.27 (m, 4H), 3.52\u2212\nDEDTC was evaluated against the solid tumor mimicking 3D\n 3.46 (m, 4H).13C NMR (126 MHz, DMSO-d6) \u03b4 215.04, 66.68,\nmulticellular spheroid model and found efficient in destructing 50.11.\nthe 3D spheroids via type I and type II PDT. 4.2.3. Synthesis of [(thpy)2-Ir-\u03bc-Cl]2. [(thpy)2-Ir-\u03bc-Cl]2 is synthe-\n sized according to the previously reported literature with slight\n4. EXPERIMENTAL SECTION modifications.66 Iridium(III) chloride (100 mg, 0.275 mmol, 1 equiv)\n 4.1. Materials and Methods. 4.1.1. Materials. All starting hydrate was dissolved in 2-ethoxyethanol/water (3:1), and 2-\nmaterials and reagents were obtained from commercial distributors (thiophen-2-yl)pyridine (107 mg, 0.68 mmol, 2.5 equiv) was added.\nand used in the synthesis as obtained. Iridium(III)chloride hydrate The reaction mixture was then allowed to reflux overnight to give a\n(IrCl3\u00b7xH2O), LysoTracker Green DND-26, and MitoTracker Green brown precipitate. The precipitate was filtered and simultaneously\nFM were obtained from Thermo Fisher Scientific. 2-(Thiophen-2- washed with ethanol and diethyl ether several times to obtain a\nyl)pyridine was purchased from BLD Pharma India. Sodium reddish-brown solid (yield 65%). 1H NMR (500 MHz, DMSO-d6) \u03b4\ndiethyldithiocarbamate and morpholine were purchased from 9.63 (d, J = 5.5 Hz, 2H), 9.31 (d, J = 5.4 Hz, 2H), 7.96 (t, J = 7.8 Hz,\nAVRA. DAPI, DPBF, and DCFH-DA were purchased from Sigma- 2H), 7.89 (t, J = 7.7 Hz, 2H), 7.75 (d, J = 7.9 Hz, 2H), 7.63 (d, J = 7.7\nAldrich. Deuterated solvents such as DMSO-d6 and CDCl3 were Hz, 2H), 7.52 (d, J = 4.8 Hz, 2H), 7.36 (dd, J = 10.3, 4.3 Hz, 2H),\npurchased from Eurisotop. Deionized water is used to perform all the 7.32 (d, J = 4.8 Hz, 2H), 7.23 (t, J = 7.4 Hz, 2H), 6.12 (d, J = 4.8 Hz,\nexperiments wherever required. The remaining chemicals were all of 2H), 5.66 (d, J = 4.8 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) \u03b4\nanalytical grade and obtained from commercial sources. 163.44, 163.00, 154.08, 152.40, 150.94, 146.53, 139.81, 138.92,\n 4.1.2. Instruments. Nuclear magnetic resonance spectra (NMR) 136.44, 135.85, 130.72, 129.42, 128.29, 120.67, 120.23, 118.36,\nwere recorded on a JEOL-500 MHz spectrometer (1H NMR: 500 118.11.\nMHz, 13C NMR: 126 MHz). UV\u2212visible absorption data were 4.2.4. General Procedure for the Synthesis of Iridium(III)\nacquired on a Shimadzu UV-2600 UV/vis/NIR spectrophotometer. Complexes. Iridium dimer (50 mg, 1 equiv) was dissolved in dry\nThe emission spectra were recorded with a Horiba Fluoromax. The DCM, and dithiocarbamate ligand (5 equiv) and Na2CO3 (10 equiv)\nfluorescence lifetimes for synthesized Ir(III) complexes were were slowly added into the solution. The reaction mixture is then\nmeasured with the help of an Edinburgh FLS 1000 (TCSPC) stirred at room temperature overnight under an inert atmosphere.\nspectrometer. The cell viability assays were performed with the help After reaction completion (confirmed by TLC), the solvent was\nof a SYNERGY H1 microplate reader. The irradiation process during evaporated under reduced pressure on the rotary evaporator. The\nphotocytotoxicity determination was conducted with the help of aqueous layer was further extracted by DCM, dried over sodium\nphotoreactor Luzchem EXPO-01 panels with blue LED (456 nm). sulfate, concentrated under reduced pressure, and then purified using\n 4.1.3. Cell Lines and Culture Maintenance. HeLa (human cervical silica gel column chromatography (1:9 ethyl acetate/hexane).\ncancer) and HCT116 (human colon cancer), human hepatic cells 4.2.4.1. Complex Ir1@DEDTC. Yellowish green solid (Yield 75%).\n 1\n(WRL-68), and PC3 (human prostate cancer) cell lines were H NMR (500 MHz, CDCl3) \u03b4 9.63 (dd, J = 6.2, 1.1 Hz, 2H), 7.86\n\n 20090 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n(d, J = 7.8 Hz, 2H), 7.75\u22127.69 (m, 2H), 7.56 (dd, J = 7.7, 1.3 Hz, DEDTC, Ir1@MORDTC, and Ir1@MEDTC complexes were excited\n2H), 7.20 (ddd, J = 7.3, 5.8, 1.5 Hz, 2H), 6.80\u22126.75 (m, 2H), 6.67 with an excitation pulse of 405 nm. The lifetime of Ir2@DEDTC and\n(td, J = 7.4, 1.3 Hz, 2H), 6.34 (dd, J = 7.8, 1.0 Hz, 2H), 3.77 (dd, J = Ir2@MORDTC was measured with an excitation pulse of a 450 nm\n13.7, 7.0 Hz, 2H), 3.53 (dd, J = 13.7, 7.0 Hz, 2H), 1.23 (t, J = 7.1 Hz, LED source. Average fluorescence lifetimes were calculated using the\n6H). 13C NMR (126 MHz, CDCl3) \u03b4 211.10, 168.82, 155.90, 151.56, best-fitting model (\u03c72 \u223c 1) with the help of the following formula for\n144.03, 135.88, 131.51, 128.85, 123.84, 122.07, 120.71, 118.44, 43.65, double exponential decay for Ir1@DEDTC, Ir1@MORDTC, and\n12.43. HRMS (ESI): calculated for C27H27IrN3S2 m/z 650.1276; Ir1@MEDTC:\nfound m/z 650.1233 [M + H]+.\n 4.2.4.2. Complex Ir1@MORDTC. Yellowish green solid (Yield A1 12 + A 2 22\n78%). 1H NMR (500 MHz, CDCl3) \u03b4 9.56\u22129.52 (m, 2H), 7.87 (d, J =\n A1 1 + A 2 2\n= 8.4 Hz, 2H), 7.77\u22127.71 (m, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.21 (t, J\n= 6.6 Hz, 2H), 6.80 (t, J = 6.9 Hz, 2H), 6.68 (t, J = 6.8 Hz, 2H), 6.32 4.5. Electron Paramagnetic Resonance (EPR). The EPR\n(d, J = 8.7 Hz, 2H), 3.92\u22123.89 (m, 2H), 3.85\u22123.81 (m, 2H), 3.70 (t, experiments were performed at room temperature (298 K) on a\nJ = 4.7 Hz, 4H). 13C NMR (126 MHz, CDCl3) \u03b4 213.15, 168.71, Bruker Biospin EMXmicro A200 spectrometer. Field modulation was\n154.91, 151.54, 143.99, 136.10, 131.51, 128.96, 123.89, 122.22, given at 100 kHz and 0.05 mT, and the microwave attenuation was\n120.91, 118.52, 66.10, 46.18. HRMS (ESI): calculated for 0.657 mW with a microwave frequency of 9.410 GHz. The complexes\nC27H25IrN3OS2 664.1068 m/z; found m/z 685.4500 [M + Na]+. were placed in a capillary positioned in a cavity. Irradiation was\n 4.2.4.3. Complex Ir1@MEDTC. Yellowish green solid (Yield 72%) executed with a 450 nm blue LED light source. The following EPR\n1\n H NMR (500 MHz, DMSO-d6) \u03b4 9.19 (d, J = 5.8 Hz, 2H), 8.20 (d, J parameters were used to acquire the spectra: sweep width of 5 mT,\n= 7.9 Hz, 2H), 7.99 (td, J = 8.1, 1.5 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 1024 points, time constant of 40.96 ms, and conversion time of 150\n7.47 (t, J = 6.6 Hz, 2H), 6.81 (t, J = 8.0 Hz, 2H), 6.67 (t, J = 8.0 Hz, ms, providing a sweep time of \u223c150 s. The spin traps TEMP, DMPO,\n2H), 6.16 (d, J = 8.5 Hz, 2H), 4.08 (s, 3H). 13C NMR (126 MHz, and BMPO (50 mM) were used to capture and identify the formation\nDMSO-d6) \u03b4 232.57, 167.84, 152.23, 151.63, 144.55, 138.09, 131.27, of different ROS produced by Ir2@DEDTC (100 \u03bcM) after\n129.31, 124.85, 123.92, 121.66, 119.72, 58.23. HRMS (ESI): irradiation with 450 nm light for 20 min.\ncalculated for C24H20IrN2OS2 m/z 609.0646; found m/z 609.0617 4.6. Cell Viability Assay. The photocytotoxicity was measured in\n[M + H]+. HeLa, HCT116, and PC3 cell lines. The cell viability assay was\n 4.2.4.4. Complex Ir2@DEDTC. Bright orange solid (yield 76%). 1H performed based on the previous light\u2212dark experiment using 3-(4,5-\nNMR (500 MHz, CDCl3) \u03b4 9.40 (dd, J = 6.4, 2.1 Hz, 2H), 7.59 (ddd, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT).69\nJ = 8.1, 7.4, 1.6 Hz, 2H), 7.48 (ddd, J = 8.1, 1.6, 0.9 Hz, 2H), 7.12 (d, Briefly, 1 \u00d7 104 cells per well were seeded in two separate 96 well\nJ = 4.8 Hz, 2H), 6.98 (ddd, J = 7.5, 5.9, 1.6 Hz, 2H), 6.21 (d, J = 4.8 plates (dark and light plates, respectively) and grown for 24 h to\nHz, 2H), 3.75 (dd, J = 14.0, 7.2 Hz, 2H), 3.52 (dd, J = 13.9, 7.2 Hz, acquire its morphology. The stock solution (5 mM) of synthesized\n2H), 1.23 (t, J = 7.2 Hz, 6H). 13C NMR (126 MHz, CDCl3) \u03b4 210.76, iridium complexes was prepared in DMSO and further diluted with\n165.20, 157.32, 151.98, 136.32, 135.74, 131.13, 127.61, 118.83, media (with FBS) so that the final concentration of DMSO in all of\n117.22, 43.51, 12.41. HRMS (ESI): calculated for C23H23IrN3S4 m/z the wells was lower than 0.4%. Cells were further treated with\n662.0404; found m/z 662.0359 [M + H]+. different concentrations (2.5, 5, 10, and 20 \u03bcM) of complex and\n 4.2.4.5. Complex Ir2@MORDTC. Bright orange solid (yield 65%). incubated for 24 h under dark conditions. After 24 h of incubation,\n1\n H NMR (500 MHz, CDCl3) \u03b4 9.32 (d, J = 5.8 Hz, 2H), 7.64\u22127.58 200 \u03bcL/well phosphate-buffered saline (PBS) was added to both\n(m, 2H), 7.49 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 4.7 Hz, 2H), 7.00 plates after removing the previous media. The light plate was then\n(ddd, J = 7.4, 5.9, 1.5 Hz, 2H), 6.20 (d, J = 4.7 Hz, 2H), 3.89 (dd, J = irradiated with an LED source (456 nm, 30 mW/cm2) for 40 min.\n10.9, 5.2 Hz, 4H), 3.70 (t, J = 4.9 Hz, 4H). 13C NMR (500 MHz, Simultaneously the dark plate was kept in the dark with PBS for 40\nCDCl3) \u03b4 212.79, 165.09, 156.31, 151.98, 136.54, 135.82, 131.04, min (without irradiation). After removing PBS, media (with FBS, 200\n127.75, 119.01, 117.30, 66.04, 46.00. HRMS (ESI): calculated for \u03bcL/well) is added in all wells and incubated for another 24 h. The\nC23H21IrN3OS4 m/z 676.0197; found m/z 676.0150 [M + H]+. cells were then washed further with PBS and incubated with a freshly\n 4.3. Quantification of 1O2 Generation (\u03d5\u0394). 1O2 was quantified prepared 10 \u03bcL (5 mg/mL) MTT in media (without FBS) for 4 h at\nusing UV\u2212vis absorbance spectroscopy with DPBF as an indicator.67 37 \u00b0C. After 4 h, 50 \u03bcL of media is removed carefully without\nDPBF assay was performed according to previously reported disturbing the formazan crystal, and 150 \u03bcL of DMSO is added to\nliterature.68 The quantum yields of 1O2 production in the presence each well. Using a microplate reader, the absorbance was measured at\nof synthesized Ir(III) complexes after photoirradiation were calculated 570 nm and cell viability was calculated by comparing the absorbance\nwith the help of degradation of the absorbance of DPBF (417 nm). of drug-treated cells with the untreated cells (cells alone). Cisplatin\nBriefly, a 50 \u03bcM solution of DPBF (alone) in DMSO is prepared, and (FDA-approved drug) is used as a positive control. All data are based\nthe absorbance at 417 nm every 5 s after irradiation with a 456 nm on three independent tests and are represented as the mean \u00b1\n(10 mW/cm2) light source was recorded. DMSO solution of Ir(III) standard error (S.E.).\ncomplex (20 \u03bcM) and DPBF (50 \u03bcM) were prepared in the dark 4.7. Detection and Quantification of Intracellular ROS.\nconditions and then irradiated at 456 nm with the same light source. Intracellular ROS levels can be detected and measured by the\nFurther, the absorbance spectra of DPBF at 417 nm were recorded fluorescent probe DCFH-DA. 5 \u00d7104 HCT116 cells per well were\nfrom 5 to 40 s of irradiation. Methylene blue (MB) was taken as a seeded and grown in 6-well plates for 24 h. The cells were treated\nreference control (\u03d5\u0394(DMSO) = 0.52). The absorbances of the complex with 20 \u03bcM iridium complex and incubated for 4 h at 37 \u00b0C, 5% CO2.\nand methylene blue are kept constant at 456 nm. The formula to Culture media was then removed, and 1 mL of PBS was added to each\ncalculate the quantum yield of 1O2 is as follows: well. Cells were subjected to an LED light source (456 nm, 30 mW/\n cm2) for 20 min. After that, cells were washed with 1\u00d7 PBS and then\n (unk)\n = (MB)\n \u00d7 (m(unk) \u00d7 F(MB))/(m(MB) \u00d7 F(unk)) incubated with DCFH-DA (20 \u03bcM) at 37 \u00b0C for 20 min under dark\n condition. Hydrogen peroxide (working conc 50 \u03bcM) was taken as a\nwhere \u03d5 denotes 1O2 quantum yield, m represents the slope of the positive control. Cells were then washed with 1\u00d7 PBS (1 mL) twice,\ngraph between absorbance changes with time, and F is the correction and then images were captured in green channel and bright field with\nfactor for absorption. the help of an Olympus fluorescent inverted microscope. All of the\n 4.4. Fluorescence Lifetime Measurement. Fluorescence life- images were taken at the same exposure time. After imaging, cells\ntimes for all the synthesized Ir(III) cyclometalated complexes were were trypsinized, collected, and centrifuged to make pellets that\nmeasured with the help of TCSPC (time-correlated single photon further dissolved in PBS. The fluorescence intensities were quantified\ncounting) equipped with a picosecond pulse diode laser excitation. A by using a microplate reader with excitation (485 nm) and emission\n20 \u03bcM solution of all the complexes was prepared in DMSO. Ir1@ (535 nm), respectively. The obtained results were plotted as a fold\n\n 20091 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nincrease in ROS generation correlated with the relative fluorescence After this, RNA (100 \u03bcg/mL) and PI (50 \u03bcg/mL) were added, and\nof DCF compared to untreated cells under dark condition. flow cytometry was performed.\n 4.8. ICP-MS Analysis. The intracellular uptake of iridium 4.15. Spheroid Formation and Imaging. HCT116 cells (2 \u00d7\ncomplexes was determined by ICP-MS. Briefly, HCT116 cells were 104 cells per well) were seeded in ultralow attachment 96-well plates\nseeded to a density of approximately 1 \u00d7 105 cells per well in tissue (U-shaped wells) and grown for 72 h to get the desired spheroids.\nculture 12-well plates. After 24 h, culture media was removed, and Spheroids were then incubated with Ir2@DEDTC (20 \u03bcM) for 24 h.\ncells were treated with Ir2@DEDTC (20 \u03bcM) for different intervals, Simultaneously, the light plate is irradiated for 40 min (30 mW/cm2)\ni.e., 2, 4, 6, and 12 h. After different incubation times with the and then incubated for another 24 h in drug free media. Spheroids\ncomplex, cells were washed (1 mL \u00d7 3) with PBS and trypsinized. were washed with 1\u00d7 PBS, and further images were captured by an\nCells were collected by centrifugation and digested with 200 \u03bcL of Olympus fluorescence inverted microscope under a bright channel for\nHNO3 (68%) for 24 h at room temperature. After 24 h of digestion in 6 days. Simultaneously, spheroids were stained with FDA/PI solution\nHNO3, the solution was diluted to a final volume of 10 mL with for 10 min in dark. Washing was done with 1\u00d7 PBS two times, and\ndistilled water or Milli-Q water. The concentration of Iridium was then images were obtained in green and red channel.\ndetermined using Agilent 8900 ICP-MS Triple Quad.\n 4.9. DAPI Staining Assay. Approximately 6 \u00d7 104 HCT116 cells\nwere seeded in six-well plates for 24 h and then further incubated with\nIr2@DEDTC for 24 h. After light irradiation, cells are washed with\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\n\n1\u00d7 PBS and then fixed with 4% paraformaldehyde for 10 min. Cells\n The Supporting Information is available free of charge at\nwere carefully washed again with 1\u00d7 PBS and permeabilized by 0.1% https://pubs.acs.org/doi/10.1021/acs.inorgchem.3c02942.\nTritonX100 for 10 min. After this, cells were washed again with 1\u00d7 studies of UV\u2212vis, NMR spectra, mass spectrometry\nPBS and incubated with DAPI (1 \u03bcg/mL in PBS) for 30 min under data, crystallographic details with bond length and bond\ndark conditions. After 30 min, cells were washed twice with 1\u00d7 PBS, angles, emission spectra, lifetime data, DPBF absorption\nand images were captured with the help of a fluorescence microscope data, photostability analysis, EPR, uptake studies for\nunder a blue channel.\n 4.10. FDA/PI Costaining. First, a stock solution of FDA (5 mg/\n iridium(III) complexes, and morphology changes in\nmL of acetone) and PI (1 mg/mL PBS) was prepared as per previous MCTS (PDF)\nliterature.70 5 \u00d7 104 cells were grown over the coverslip in a 6-well Accession Codes\nplate for 24 h. The cells were then treated with 20 \u03bcM Ir2@DEDTC CCDC 2277278 and 2277282 contain the supplementary\nfor 6 and 24 h. After completion of the incubation time, the medium\nwas replaced with PBS for the light group and irradiated for 20 min.\n crystallographic data for this paper. These data can be obtained\nCells were carefully washed with 1\u00d7 PBS and incubated with FDA free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by\n(working concentration: 8 \u03bcg/mL) and PI (2 \u03bcg/mL) for 10 min in emailing data_request@ccdc.cam.ac.uk, or by contacting The\nthe dark. Then, cells were washed slowly with 1\u00d7 PBS twice, and then Cambridge Crystallographic Data Centre, 12 Union Road,\ncoverslips were mounted over the slides to capture the images using Cambridge CB2 1EZ, UK; fax: +44 1223 336033.\nan Olympus fluorescence microscope under bright field and green and\nred channels.\n 4.11. Fluorescence Imaging and Cellular Uptake. HCT116\ncells (6 \u00d7 104 cells per well) were seeded in 6-well plates and grown\n \u25a0 AUTHOR INFORMATION\n Corresponding Author\nover a coverslip for 24 h. The cells were then treated with iridium(III) V. Venkatesh \u2212 Department of Chemistry, Indian Institute of\ncomplex and incubated for 8 h. After removing media, cells were Technology Roorkee, Roorkee, Uttarakhand 247667, India;\ncarefully washed with 1\u00d7 PBS and coverslips were mounted and orcid.org/0000-0001-9520-6842; Email: venkatesh.v@\nimages were obtained using an Olympus fluorescence microscope cy.iitr.ac.in\nunder bright light and red channel. The images reported were then\nfurther processed using ImageJ software. Authors\n 4.12. Cellular Localization. Briefly, HCT116 cells (6 \u00d7 104 cells Monika Negi \u2212 Department of Chemistry, Indian Institute of\nper well) were seeded in six-well plates and grown for 24 h. Cells were Technology Roorkee, Roorkee, Uttarakhand 247667, India\nthen incubated with Ir2@DEDTC (20 \u03bcM) for 6 h. After 6 h, cells Tejal Dixit \u2212 Department of Chemistry, Indian Institute of\nwere washed with 1\u00d7 PBS and incubated with Mitotracker Green Technology Roorkee, Roorkee, Uttarakhand 247667, India\n(500 nM), Lysotracker Green (500 nM), and DAPI (1 \u03bcg/mL),\nrespectively, for 30 min in the dark. After this, cells were carefully Complete contact information is available at:\nwashed with 1\u00d7 PBS twice, and images were captured with the help of https://pubs.acs.org/10.1021/acs.inorgchem.3c02942\nOlympus fluorescence microscope. The images were further\nprocessed with Fiji imageJ software and Pearson\u2019s R value is Author Contributions\ndetermined using Coloc2 plugin. V.V. and M.N. conceptualized the study. M.N. synthesized and\n 4.13. JC-1 Staining. HCT116 cells (5 \u00d7 104 cells per well) were characterized the complexes and performed all the biological\nseeded in 12-well plates and grown for 24 h. Cells were then studies. T.D. performed and analyzed the single crystal XRD\nincubated with Ir2@DEDTC (20 \u03bcM) for 6 h. After 6 h, the cells analysis of the synthesized complexes. The manuscript was\nwere washed with 1\u00d7 PBS and incubated with JC-1 (5 \u03bcg/mL) for 30\n written through the contributions of all authors. All authors\nmin in the dark. After this, cells were carefully washed with 1\u00d7 PBS\ntwice, and images were captured with the help of an Olympus have approved the final version of the manuscript.\nfluorescence microscope under bright field, green, and red channels Notes\nwith an exposure time of 394.4 ms. The authors declare no competing financial interest.\n 4.14. Cell Cycle Arrest. HCT116 cells (6 \u00d7 104 cells per well)\nwere seeded in 12 well plates and grown for 24 h. Cells were then\nincubated with Ir2@DEDTC (20 \u03bcM) for 24 h. After 24 h, the light\nplate is irradiated for 40 min (30 mW/cm2) and then incubated for\n \u25a0 ACKNOWLEDGMENTS\n V.V. sincerely acknowledges the Science and Engineering\nanother 24 h. Cells were washed with 1\u00d7 PBS and trypsinized to Research Board (CRG/2020/001398), New Delhi, India. The\nobtain the pellet. After this, all the pellets were carefully washed with authors are thankful to Dr. B. V. V. S. Pavan Kumar for\nice-cold 1\u00d7 PBS and further 70% ethanol was added dropwise while providing access to the fluorescence microscope facility. We\nvortexing the pellets and incubated for 2 h at 4 \u00b0C to fix the cells. are also grateful for Dr. Reena Kumari for her suggestions\n 20092 https://doi.org/10.1021/acs.inorgchem.3c02942\n Inorg. Chem. 2023, 62, 20080\u221220095\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nwhile performing biological assays. We acknowledge the Iridium(III)-Naphthalimide Complex: Toward Highly Enhanced\nInstitute Instrumentation Centre (IIC), IIT Roorkee for Hypoxia-Inducible Factor-1. ACS Appl. Bio Mater. 2020, 3, 252\u2212262.\nEPR, ICP-MS and FLS facilities, DST-FIST for NMR and (18) Fan, Z.; Rong, Y.; Sadhukhan, T.; Liang, S.; Li, W.; Yuan, Z.;\n Zhu, Z.; Guo, S.; Ji, S.; Wang, J. Single-Cell Quantification of a Highly\nsingle crystal XRD facility (SR/FST/CS-II/2018/72(C).\n Biocompatible Dinuclear Iridium(III) Complex for Photocatalytic\n\n\u25a0 REFERENCES\n (1) Sawant, S.; Shegokar, R. Cancer research and therapy: where are\n Cancer Therapy. Angew. Chem., Int. Ed. 2022, 61, e202202098.\n (19) Huang, H.; Banerjee, S.; Sadler, P. J. Recent advances in the\n design of targeted iridium(III) photosensitizers for photodynamic\nwe today. Int. J. Cancer Ther. Oncol. 2014, 2, 020408. therapy. ChemBioChem. 2018, 19, 1574\u22121589.\n (2) Imberti, C.; Zhang, P.; Huang, H.; Sadler, P. J. 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