Cyclometalated Ir(III) theranostic molecular probe enabled mitochondria targeted fluorescence-SERS-guided phototherapy in breast cancer cells.

{"full_text": " Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Photochemistry & Photobiology, B: Biology\n journal homepage: www.elsevier.com/locate/jphotobiol\n\n\n\n\nCyclometalated Ir(III) theranostic molecular probe enabled mitochondria\ntargeted fluorescence-SERS-guided phototherapy in breast cancer cells\nChandana Reghukumar a, b, Shanmughan Shamjith a, b, Vishnu Priya Murali a,\nPilankatta K. Ramya a, b, Kokkuvayil Vasu Radhakrishnan a, b, Kaustabh Kumar Maiti a, b, *\na\n Chemical Sciences & Technology Division (CSTD), Organic Chemistry Section, CSIR-National Institute for Interdisciplinary Science & Technology (CSIR-NIIST),\nIndustrial Estate, Pappanamcode, Thiruvananthapuram 695019, Kerala, India\nb\n Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: The increased energy demands inherent in cancer cells necessitate a dependence on mitochondrial assistance for\nMitochondria targeting their proliferation and metastatic activity. Herein, an innovative photo-medical approach has been attempted,\nPhotodynamic therapy specifically targeting mitochondria, the cellular powerhouses, to attain therapeutic benefit. This strategy facil\u00ad\nIridium complex\n itates the rapid and precise initiation of apoptosis, the programmed cell death process. In this goal, we have\nReactive oxygen species\n synthesized cyclometalated Iridium (III) molecular probes, denoted as Ir-CN and Ir-H, with a nitrile (CN) and a\nCancer theranostics\nSERS hydrogen-functionalized bipyridine as ancillary ligands, respectively. Ir-CN has shown superior photosensitizing\n properties and lower dark cytotoxicity compared to Ir-H in the breast cancer cell line MCF-7, positioning it as the\n preferred probe for photodynamic therapy (PDT). The synthesized Ir-CN induces alterations in mitochondrial\n membrane potential, disrupting the respiratory chain function, and generating reactive oxygen species that\n activate signaling pathways leading to cell death. The CN-conjugated bipyridine ligand in Ir-CN contributes to\n the intense red fluorescence and the positive charge on the central metal atom facilitates specific mitochondrial\n colocalization (colocalization coefficient of 0.90). Together with this, the Iridium metal, with strong spin-orbit\n coupling, efficiently generates singlet oxygen with a quantum yield of 0.79. Consequently, the cytotoxic\n singlet oxygen produced by Ir-CN upon laser exposure disrupts mitochondrial processes, arresting the electron\n transport chain and energy production, ultimately leading to programmed cell death. This mitochondrial\n imbalance and apoptotic induction were dually confirmed through various apoptotic assays including Annexin V\n staining and by mapping the molecular level changes through surface-enhanced Raman spectroscopy (SERS).\n Therefore, cyclometalated Ir-CN emerges as a promising molecular probe for cancer theranostics, inducing laser-\n assisted mitochondrial damage, as tracked through bimodal fluorescence and SERS.\n\n\n\n\n1. Introduction instances [4]. Subsequently, as an alternative anti-cancer therapy,\n photodynamic therapy (PDT) is being utilized in certain cancer treat\u00ad\n According to WHO data, cancer accounts for one in six leading causes ments which is a light-based technology that has benefits over conven\u00ad\nof death worldwide and is the second biggest cause of mortality [1,2]. tional cancer therapies for solid tumors [5].\nOver time, there has been a sharp rise in the incidence of cancer, which PDT has a superior treatment option to traditional cancer treatment\nis still a hazard. Cancer is the term used to describe the unchecked modalities as an individualized precision treatment by its precise\nproliferation of cells, often resistant to the anti-growth mechanism [3]. controllability and minimal/non-invasiveness [6]. PDT is the result of\nThese cancer cells can multiply, evade apoptosis, create new blood the interaction of three key components: a photosensitizer (PS), light\nvessels, and spread throughout the body. Due to its adverse side effects, with a certain wavelength, and the presence of molecular oxygen [7].\ndrug resistance, and non-targeted action, chemotherapy, the traditional The photosensitizer absorbs light when exposed to the target tissue,\nmedication-based treatment, was often unsuccessful in a majority of which sets off a cascade of photochemical events that lead to the\n\n\n\n * Corresponding author at: Chemical Sciences & Technology Division (CSTD), Organic Chemistry Section, CSIR-National Institute for Interdisciplinary Science &\nTechnology (CSIR-NIIST), Industrial Estate, Pappanamcode, Thiruvananthapuram 695019, Kerala, India.\n E-mail address: kkmaiti@niist.res.in (K.K. Maiti).\n\nhttps://doi.org/10.1016/j.jphotobiol.2023.112832\nReceived 9 October 2023; Received in revised form 8 December 2023; Accepted 15 December 2023\nAvailable online 21 December 2023\n1011-1344/\u00a9 2023 Elsevier B.V. All rights reserved.\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\nproduction of ROS [8]. To achieve the desired therapeutic effect, a va\u00ad The exclusive capacity of Ir-CN to generate singlet oxygen upon laser\nriety of factors should be considered when standardizing the treatment irradiation endows it with an ideal profile for precision therapy. Beyond\nprotocol, including the type of PS used, the dose administered, the its phototherapeutic potential, Ir-CN boasts a dual functionality as a\nintracellular location of the PS upon internalization, the drug-to-light two-in-one bimodal probe. The inherent red fluorescence and the\ninterval (DL1), the total amount of light applied, its wavelength, and distinct Raman peak associated with the CN moiety enable its utility in\nfluence rate, the characteristics of the tumor, and the local availability of mitochondria-targeted image-guided photodynamic therapeutic assess\u00ad\noxygen [6,9,10]. ment. This as-mentioned approach leverages bimodal fluorescence and\n Considering the therapeutic outcome of cisplatin, other transition Raman spectroscopy for precise monitoring of the therapeutic progres\u00ad\nmetal complexes have been investigated as anti-cancer agents by rede\u00ad sion, highlighting the multifaceted utility of Ir-CN in scientific research\nsigning by ligand substitution on existing chemical structures or by and potential clinical applications. (Scheme 1).\nsynthesizing an entirely new chemical entity [11,12]. Likewise, the\nusage of metal complexes as photosensitizers for PDT has dramatically 2. Results and Discussion\nincreased over the past decade [13]. The fact that the metal complexes\nsatisfy several crucial parameters for a photosensitizer is the cause of 2.1. Design, Synthesis, and Characterization of the Cyclometalated Ir(III)\ntheir increasing popularity. The heavy atom promotes spin-orbit Complexes\ncoupling, leading to a population of efficient and ultrafast triplet-\nexcited states [14,15]. In general, high yields of triplet excited states Cyclometalated Iridium (III) complexes, Ir-CN and Ir-H, were syn\u00ad\nlead to high yields of singlet oxygen generation. Nonetheless, due to thesized through a multi-step procedure. Initially, a chlorine-bridged\ntheir prolonged emission lifetimes, metal complexes are sensitive to the dimeric Iridium complex was formed by employing the Nonoyama re\u00ad\nintracellular milieu, making them potential \u201csee and cure\u201d agents [16]. action, which involved the reaction between IrCl3\u22c53H2O and 2-phenyl\u00ad\nMetal complexes are typically photostable under sustained one- and pyridine. Subsequently, ancillary ligands, namely 4,4\u2032-([2,2\u2032-\ntwo-photon illumination, in contrast to the majority of colored organic bipyridine]-4,4\u2032-diyl) dibenzonitrile (for Ir-CN) and 4,4\u2032-diphenyl 2,2\u2032-\ncompounds. This allows for the longer recycling of the PS and, as a bipyridine (for Ir-H), were synthesized via a palladium-catalyzed Suzuki\nresult, a reduction in the overall dose of PS required [17]. The relative cross-coupling reaction [28]. Finally, the chlorine bridges were replaced\nease with which metal complexes can be created, where various ligands with bipyridine ligands, yielding Ir-CN and Ir-H as solid compounds with\nand metal centers can be coupled in an almost combinatorial fashion, orange and pale orange colors, respectively. To substitute the chlorine\nadding to these desirable photophysical properties, provides the chance counter ions in the complexes with hexafluorophosphate anions, the\nto modify those features as needed [18]. anion exchange reaction was carried out using potassium hexa\u00ad\n In the scenario of intracellular metabolic events, mitochondria serve fluorophosphate. Comprehensive characterization of the prepared\nas both the location of eukaryotic oxidative metabolism and the source cyclometalated iridium complexes Ir-CN, Ir-H, and their intermediates\nof energy as reflected in the production of ATP [19]. The tricarboxylic was accomplished using advanced spectroscopic techniques, including\n 1\nacid cycle and oxidative phosphorylation, which correspond to the H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS).\nsecond and third stages of aerobic respiration, are used to characterize (Fig. S1 to S11).\nthe common mechanism of mitochondrial oxidation [20]. Contrarily, Iridium complexes exhibit a range of advantageous characteristics\nmitochondria are essential for controlling intrinsic apoptosis. The when employed as photosensitizing agents in Photodynamic Therapy\nintermembrane space (IMS) of mitochondria releases proproteins of (PDT). These complexes possess unique photophysical properties, such\napoptosis to the cytosol in the normal apoptotic route [21]. The ability as prolonged excited states and efficient inter-system crossing, making\nof cancer cells to switch between glycolysis and oxidative phosphory\u00ad them exceptionally proficient at generating reactive oxygen species\nlation has been found to have a role in providing resistance towards when exposed to light. Moreover, their outstanding stability in biolog\u00ad\noncogene inhibition [22,23]. Since it was also discovered that mito\u00ad ical environments and versatile chemical adaptability further enhance\nchondria play a significant role in controlling cell death, mitochondria- their suitability as PDT agents. These features hold the potential for\ntargeted photodynamic therapy (Mt-PDT) was developed as a promising precise and selective eradication of cancer cells while minimizing\nstrategy for eliminating cancer cells [21,24]. Many mitochondrial tar\u00ad damage to healthy tissues. In both complexes, the central metal\ngeting molecular PSs are reported using lipophilic cations in line with component of the cyclometalated ligand imparts a net +1 charge to the\nthe overall mitochondrial transmembrane potential (MTP) [25]. In overall complex, enhancing its specific interaction capacity with nega\u00ad\ngeneral, cyclometalated Ir (III) complexes are well-known examples of tively polarized mitochondria, making it an excellent candidate for\nmitochondrial localization due to their inherent positive charge on the targeting mitochondria in PDT. The CN centers within the appended\nmetal, making it an ideal Mito-targeted PDT agent [26,27]. Considering bipyridine portion of Ir-CN introduce a strong electron-withdrawing\nall these facts, we hypothesized that cyclometalated Iridium (III) com\u00ad effect, resulting in an extended red emission in the compound. Addi\u00ad\nplexes can be utilized as efficient molecular probes for creating new tionally, the presence of a triple bond in the CN unit imparts distinct\nluminous PDT agents. Raman fingerprints in the cell\u2019s silent region of Raman spectra, resem\u00ad\n Considering this in mind, we have demonstrated a comprehensive bling a marker peak for Raman-based characterization of the probe\nexploration encompassing the design, synthesis, and meticulous opti\u00ad when compared to the non-substituted counterpart Ir-H. The disparities\nmization of two distinct cyclometalated Iridium complexes, denoted as in emission profiles and the sharp Raman peak at 2190 cm\u2212 1 serve as\nIr-CN and Ir-H, both of which exhibit remarkable pH stability and valuable distinguishing features for these molecules in both fluorescence\nphotostability. Our investigation extends to the characterization of these and Raman-based platforms (Fig. 1d, e).\ncomplexes, unveiling their potential as photosensitizers in various ap\u00ad\nplications. The rigorous scrutiny of the photophysical properties and 2.2. Energetic Assessment of Ir-CN and Ir-H\ncomprehensive toxicity profiling underscores the clear superiority of Ir-\nCN over Ir-H. This observation renders Ir-CN an exceptionally promising Head-Gordon and colleagues\u2019 density functional theory (DFT) tech\u00ad\ncandidate for further in-depth in vitro assessment. Remarkably, Ir-CN nique using the wB97xD method is used for the optimization of all ge\u00ad\ndemonstrates swift internalization into cancer cells, achieving localiza\u00ad ometries, which integrates dispersion and has excellent geometry\ntion within mitochondria in a time frame of less than one hour. optimization performance. As the molecular complexes are large, the 6-\nFurthermore, it exerts significant antitumor activity when subjected to 31G(d) moderate size double zeta split-valence basis set with polar\u00ad\nlaser irradiation, primarily through the activation of apoptotic pathways isation function is used for all main block elements, while the LanL2DZ\ndriven by an abnormal amplification of reactive oxygen species (ROS). basis set is used for Ir, with the inner core electrons replaced with Hay\n\n 2\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\n\n\nScheme 1. Schematic illustration of mitochondrial-targeted PDT induced by Ir-CN (Created with BioRender.com). Ir-CN, the novel Ir(III) based photosensitizer, upon\nincubation with MCF-7 breast cancer cells, gets easily internalized to provide red fluorescence mainly from the mitochondrial region of the cells. The Ir-CN treated\ncells were irradiated with a laser to induce ROS generation, leading to cellular apoptosis.\n\n\nand Wadts\u2019 relativistic effective core potential (ECP). This method is width 3 nm) (Fig. S12). Moving on to the Raman spectroscopic analysis,\nabbreviated as wB97xD/BS. Furthermore, at the wB97xD/BS level, the complex Ir-CN shows a sharp Raman peak at 2190 cm\u2212 1 (Fig. 1d)\nvibrational frequency analysis was conducted to validate the nature of corresponding to the CN vibration in bare Raman mode making it an\nthe optimized structures. The minimum of the complexes was verified by ideal probe for intracellular investigation through Raman modality\nensuring the absence of imaginary frequencies. To account for solvation whereas the absence of this peak in the Ir-H complex clearly confirms the\neffects, particularly in dimethylsulfoxide (DMSO), the optimization presence of CN. Additionally, the SERS studies of Ir-CN were carried out\nprocess incorporated the implicit self-consistent reaction field model using spherical gold nanoparticles (AuNPs: 35\u201340 nm) to evaluate the\n(SCRF), specifically SMD, as implemented in Gaussian16. The band gaps surface-enhanced Raman peaks of the Ir-CN for monitoring the ultra\u00ad\n(Eg) for both complexes are determined by subtracting the correspond\u00ad sensitive responses. The Raman fingerprinting of Ir-CN showed excellent\ning HOMO and LUMO energies. It is worth noting that the complex Ir-H Raman signals, viz., highly intense peak 2129 cm\u2212 1 due to the CN vi\u00ad\nexhibits a slightly higher Eg compared to the Ir-CN complex. The ener\u00ad bration and few sharp bands such as the one corresponding 1023 cm\u2212 1\ngetics of the two complexes are tabulated in Table 1. due to ring breathing from the phenyl part in the complex, making them\n promising candidates for SERS based probes for imaging the cells\n through Raman platform (Fig. 1e). Utilizing pH titration at several pH\n2.3. Photophysical and Stability Evaluation of Ir-CN and Ir-H\n levels ranging from 4.5 to 9.5 confirms the stability of Ir-CN and Ir-H\n absorption and emission characteristics in a range of pH environments\n The UV\u2013Vis absorption of both the complexes has been measured in\n (Fig. 1f and S14).\nDMSO (10 \u03bcM at 25 \u25e6 C). UV\u2013Vis absorption spectrum of the complexes\n Assessing photostability is a critical consideration when examining\nIr-CN (10 \u03bcM in DMSO at 25 \u25e6 C) showed energy absorption bands at\n photosensitizing agents for photodynamic therapy (PDT). To assess this,\n230\u2013350 nm (Fig. 1c) corresponding to the spin-allowed ligand centered\n both the intricate Ir-CN and Ir-H complexes were subjected to exposure\n\u03c0-\u03c0* transitions for cyclometalated (Ppy) and ancillary ligands [29]. The from a 405 nm laser over a specific duration, and their photophysical\nmixed singlet and triplet metal-to-ligand charge transfer (1MLCT and\n3 properties were thoroughly analyzed. As anticipated, both complexes\n MLCT) and ligand-to-ligand charge-transfer (LLCT) transitions are\n exhibited remarkable stability over an extended timeframe, providing\nresponsible for the weak and low-energy bands between 400 and 510\n substantial evidence in favor of the photostability of these molecules (as\nnm16. When the complexes are subjected to long UV light irradiation\n depicted in Fig. 1g, h, S13). Furthermore, when evaluating the param\u00ad\nfrom a standard UV lamp, both emanate sharp red fluorescence. The\n eters within environments comprising 10% fetal bovine serum (FBS),\ncomplex Ir-CN exhibits a wide emission peak centered at 645 nm after\n phosphate-buffered saline (PBS), and pure water, we observed consis\u00ad\nbeing excited at 405 nm (excitation slit width 3 nm, emission slit width\n tent absorption and emission intensity profiles. This once again un\u00ad\n3 nm) (Fig. 1c) while Ir-H showed a wide emission peak centered at 620\n derscores the robust and enduring nature of our complex.\nnm upon excitation at 385 nm (excitation slit width 3 nm, emission slit\n\n 3\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\n\n\nFig. 1. (a) Schematic workflow of synthesis of Ir-CN. (b) DFT optimized structure of Ir-CN (c) UV\u2013Vis absorbance and emission spectra of 10 \u03bcM Ir-CN in DMSO at\n25 \u25e6 C (d) Characteristic Raman Fingerprint of Ir-CN (e) SERS spectra of Ir-CN with spherical gold nano-particle in 1:9 ratio indicating the cell silent peak at 2129\ncm\u2212 1 (f) Graph showing the stability of Ir-CN in varying pHs (g) Graph depicting the photostability of Ir-CN without laser and (h) with laser. (For interpretation of the\nreferences to colour in this figure legend, the reader is referred to the web version of this article.)\n\n\n\nTable 1\nThe energy data (in a.u) obtained at wB97xD/SMD/BS level for the Ir-CN and Ir-H.\n Molecules SCF energy Zero-point corrected energy Free energy Free energy correction HOMO energy LUMO energy Band gap\n\n E ZPE G G(corr) EHOMO ELUMO Eg\n\n Ir-CN \u2212 2203.48478 \u2212 2202.83258 \u2212 2202.91028 0.574494 \u2212 0.27766 \u2212 0.02785 0.24982\n Ir-H \u2212 2019.06802 \u2212 2018.41332 \u2212 2018.48589 0.582132 \u2212 0.27652 \u2212 0.01960 0.25691\n\n\n\n2.4. Photoactivation and Singlet Oxygen Generation of Ir-CN and Ir-H (Fig. 2c) and a structurally significant molecule [Ru(Bpy)3]Cl2 as stan\u00ad\n dard reference material, the Ir-CN and Ir-H were examined for their\n Although, compared to organic photosensitizers, the use of metal- ability to generate 1O2. For this, the material under investigation was\nbased complexes for phototherapy is still in its infancy, the chemical mixed with oxygen scavenger DPBF in a 1:3 ratio of its absorbance. The\nexpertise gained through years of experience with cisplatin and other mixture was capped well and irradiated with a 405 nm laser, later its\nmetal-based medications can speed up the development of photo\u00ad absorption and emission profiles were measured. The molecules when\nactivatable complexes [30]. Complexes based on noble metals display darted with laser sharply attenuate the absorption of DPBF at 410 nm\nPDT activity as photosensitizers due to their tunable photophysical and due to the singlet oxygen cycloaddition (Fig. 2d). Similarly, the sharp\nphotochemical features and considerable ROS generation [11]. In emission bands of DPBF at 450 and 500 nm were quenched effectively,\nconsequence, advancements in this field open new opportunities for the which would also correlate with the singlet oxygen production (Fig. 2e).\nuse of bioinorganic chemistry in medicine. In general, because of their The ability of Ir-CN to generate 1O2 is amply demonstrated by a decrease\nadjustable photophysical and photochemical properties, cyclometalated in the absorbance and emission profiles of DPBF within 240 s. When\niridium complexes have recently attracted a lot of attention as potential compared to the reference [Ru(Bpy)3]Cl2, which had a singlet oxygen\nPDT agents. It is well known that the efficiency of intersystem crossover quantum yield of 0.57, the Ir-CN was found to have a value of 0.79\n(S1 to T1) and the subsequent energy transfer from triplet oxygen are whereas Ir-H has a value of 0.72 making Ir-CN a good candidate over Ir-\ndirectly correlated with 1O2 generation (Fig. 2a). These generated oxy\u00ad H (Fig. 2f, S15).\ngen species attack the organelles leading to apoptosis (Fig. 2b). Using the The increased quantum yield is clearly determined by the liner plot\nwell-known 1O2 scavenger dye 1,3-diphenylisobenzofuran (DPBF) of the change in absorbance of DPBF (\u0394A) caused by Ir-CN and Ir-H with\n\n\n 4\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\n\n\nFig. 2. (a) Schematic representation of Jablonski diagram depicting Type II photosensitization and singlet oxygen generation (b) Pictorial representation of apoptosis\ninduced by singlet oxygen generation in cells (c) Mechanism of emission turn-off of DPBF and turn-on of SOSG during singlet oxygen cycloaddition (d) Attenuation of\n410 nm peak of DPBF during photoactivation of Ir-CN (e) Emission quenching of DPBF with generated singlet oxygen during the photoactivation of Ir-CN (f) Linear\nplot of Ir-CN and Ru(Bpy)3Cl2 used for the calculation of singlet oxygen quantum yield (g) Relative singlet oxygen generation of Ir-CN along with other combination\n(h) Confirmation of singlet oxygen generation ability of Ir-CN and other combination using SOSG (A- SOSG alone, B- SOSG + Ir-CN without laser, C- SOSG+ Ir-CN\nwith laser irradiation for 1 min, D- SOSG+ Ir-CN with laser irradiation for 2 min).\n\n\nrespect to time. Both DPBF alone and in different combinations are oxygen during the photoirradiation process is amply confirmed by the\nunable to have this effect. The relative singlet oxygen production fast regeneration of SOSG emission (Fig. 2h).\ncalculated by the change in optical density of DPBF with various trials\nevidentially confirms the superiority of Ir-CN over other combinations.\nThese sharp emission and absorption changes caused by Ir-CN with the 2.5. Evaluation of Cytotoxicity and Cellular Internalization of Molecular\nlaser to the scavenger molecule unequivocally supported Ir-CN\u2019s po\u00ad Probe Ir-CN and Ir-H\ntential as a photosensitizer moiety (Fig. 2g). Finally, the singlet oxygen\nproduction in the solution state was further confirmed by the well- The assessment of cytotoxicity is a prerequisite for any molecular\nknown singlet oxygen indicator Singlet Oxygen Sensor Green (SOSG). probe before proceeding to further studies on in vitro biological\nThe PeT phenomenon between the anthracene subunit and the central assessment. Being non-toxic to the cells in dark conditions is an imper\u00ad\noxygen atom in the main ring quenched the dye SOSG\u2019s emission ative feature for any PDT agent, at the same time, it should be activated\nproperties. Upon interaction with the singlet state oxygen, the anthra\u00ad upon proper irradiation. MTT assay was employed to determine the dose\ncene part underwent a cycloaddition reaction which eventually stops the and time-dependent cytotoxicity of Ir-CN and Ir-H in the absence of laser\nPeT process and turns on the emission behavior. The dye SOSG alone irradiation. The assay enables the measurement of cell viability by\nand in combination with the complex Ir-CN will emit light with a rela\u00ad assessing the cleavage of tetrazolium salt by mitochondrial dehydroge\u00ad\ntively weak center of emission at 525 nm, but after being exposed to 405 nase. Initially, MCF-7, a breast cancer cell line was subjected to the\nnm laser radiation for up to two minutes, the emission profile will treatment with both the candidate compounds. The results revealed that\ndramatically rise by 4 to 6 times (Fig. 2h, D). The presence of singlet even at a concentration as low as 5 \u03bcM, Ir-H caused a depletion in cell\n viability to 60% (Fig. S23, Table S2), whereas Ir-CN does not exhibit a\n\n 5\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\nsignificant cytotoxic effect up to 20 \u03bcM concentration within 6 h, Thus 10 \u03bcM concentration of Ir-CN for 60 min incubation was chosen for\nshowing 98% cell viability (Fig. S24, Table S3). As shown in Fig. S25, all the further experiments. With this optimized internalization time and\neven after 24 h incubation, 10 \u03bcM concentration of the latter retained concentration, Ir-CN uptake by MCF-7 cells was again confirmed using\nabout 90% cell viability (Table S4). SERS analysis. Upon SERS imaging, Ir-CN specific peaks (1023 cm\u2212 1,\n Owing to the augmented dark cytotoxic nature, Ir-H was subse\u00ad 2129 cm\u2212 1) were observed from the average scan spectra of the treated\nquently eliminated and Ir-CN was chosen for further studies. Further\u00ad cells (Fig. S18 a, b, c) as compared to the untreated control cells which\nmore, Ir-CN was also found minimal cytotoxic effect on the normal only produced the cell peaks (Fig. S18 d, e, f).\nbreast epithelial cell line MCF-10-A as well (Fig. S26). Since, 10 \u03bcM\nconcentration was used for the photophysical studies and has shown no 2.6. Assessment of Mitochondria Targeting Ability of Ir-CN\nconsiderable dark toxicity in both the normal and cancer cells, the same\nconcentration was maintained for the time-dependent cellular inter\u00ad MCF-7 cells were treated with Ir-CN with the optimized concentra\u00ad\nnalization studies. tion of 10 \u03bcM followed by mitotracker green, lysotracker green, and\n Important variables of the pharmacokinetic and pharmacodynamic nuclear targeting Hoechst as per the pre-optimized condition for\ncharacteristics of medicines and therapeutic candidates include suc\u00ad colocalization experiment. The red fluorescence of Ir-CN and green\ncessful cellular uptake and membrane permeability. The innate fluo\u00ad fluorescence from the lyso and mitotrackers and blue fluorescence from\nrescence property renders an added advantage of tracking the fate and Hoechst-33,342 were subjected to superimposition. Ir-CN was found to\nactivity of the molecule within the cell without the aid of a tracking be superimposed well with the green fluorescence of the mitotracker as\nreagent. Cellular internalization of our candidate molecule Ir-CN at shown in Fig. 4a, with a Pearson coefficient value of 0.9, which predi\u00ad\nthree different time points (30 min, 60 min, and 120 min) was moni\u00ad cates the fact that the probe is targeting mitochondria. In the case of the\ntored by visualizing the fluorescence emitted from MCF-7 cells treated nuclear and lysotrackers, only 0.6 and 0.54 were obtained as their\nwith it. Though the compound was seen successfully internalizing from respective R values, indicating minimal targeting of the probe molecules\n30 min of incubation onwards, maximum fluorescence intensity was towards those organelles (Fig. 4).\nobserved with 60 min incubation, and no further increase was seen even\nwith 120 min of incubation. Therefore, 60 min was fixed as the optimal\ntime of internalization for further experiments (Fig. 3b, c). Besides, the 2.7. Assessment of the PDT Effects of Ir-CN in MCF-7 Cells\nconcentration-dependent cellular internalization studies demonstrated\nthat the probe fluorescence is visible even at a concentration of 5 \u03bcM but The excitation of the photosensitizers produces abundant amounts of\nless intense as compared to 10 \u03bcM or 15 \u03bcM concentrations (Fig. 3d, f). singlet oxygen, a striking feature exploited in PDT. Numerous anti-\n cancer drugs are also known to generate singlet oxygen, which in turn\n\n\n\n\nFig. 3. (a) A pictorial representation of internalization of Ir-CN into MCF-7 cells. Standardization of concentration and duration of exposure to Ir-CN via (b) time-\ndependent cellular internalization studies (c) graph showing fluorescence intensity at different durations of incubation with the complex (d) concentration-dependent\ncellular internalization studies (e) graph depicting fluorescence intensity upon treatment with different concentrations of the complex. (scale bar = 50 \u03bcM).\n\n 6\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\n\n\nFig. 4. Colocalization studies for the precise identification of the organelle to which Ir-CN is localized within the cell using (a) Mito tracker green, (b) Nuclear tracker\n(Hoechst) and (c) Lyso tracker green (Scale bar = 50 \u03bcm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version\nof this article.)\n\n\nacts as \u201cpoison\u201d towards the cells, causing significant depletion in cell expressed on the surface of the apoptotic cells to provide the green\nviability. [31] fluorescence of FITC. Co-staining with PI enabled the segregation of the\n MTT assay performed with the Ir-CN treated, laser irradiated (405 cells in early apoptosis with more FITC fluorescence and less PI fluo\u00ad\nnm) cells showed a profound cytotoxic effect with a cell death rate of rescence (Q4) and late apoptotic cells with both FITC and PI fluores\u00ad\n81%. Whereas, the Ir-CN-treated non-irradiated cells and untreated cence(Q2). Healthy cells are least stained by both dyes (Q3) while the\nlaser-exposed cells showed only about 10% and 3.9% cytotoxicity necrotic cell population has the maximum PI fluorescence (Q1). Here we\nrespectively. The cytotoxic response brought by Ir-CN PDT was found to observed >50% of cells in the early apoptotic stage of Ir-CN + laser-\nbe better than that of Doxorubicin-induced cytotoxicity at the same treated cells meanwhile the probe-alone treated cells and untreated cells\nconcentration of 10 \u03bcM within 24 h time (Fig. 5b). Thus, it is evident that + laser groups showed only 4.7% and 10% cells in the same quadrant\nlaser irradiation activates the Ir-CN molecules internalized by the cells (Q4) respectively with the maximal distribution of the cells in healthy\nand triggers ROS-mediated cell death. The ROS generation was analyzed cell quadrant (Q3). A small population of cells in Ir-CN + laser-treated\nby fluorescent imaging and flow cytometric analysis using DCFH-DA group was also found to be in a late apoptotic state as well, emphasizing\nstaining assay [32] (Supporting information 3.4, Fig. S18). the PDT-mediated apoptosis-inducing effect of Ir-CN (Fig. 5c and d).\n Further, cell staining apoptotic assays were performed to conclude\n the cell death-inducing effect of Ir-CN-PDT. For this, Acridine orange-\n2.8. Evaluation of Programmed Cell Death Phenomenon by Ir-CN ethidium bromide dual staining was conducted to visualize orange-red\n fluorescing apoptotic cells. The assay exploits the ability of the dyes to\n It is a well-known fact that irradiation of photosensitized cells leads cross the cell membrane. Acridine orange can diffuse into intact cell\nto heightened light-catalyzed singlet oxygen generation in close prox\u00ad membranes and fluoresce green whereas ethidium bromide is able to\nimity to the subcellular location of photosensitizer binding. The cross only the compromised cell membranes such as those of necrotic or\nphototoxicity in the cells is predominantly mediated by the rapid initi\u00ad apoptotic cells [35,36]. Thus, the live cells, with acridine orange alone,\nation of apoptosis, a very complex, multi-step, multi-pathway cell-death fluoresce green while the dead cells and those with a compromised\nprocess. Tumor cells exhibit the tendency to resist apoptotic progression membrane fluoresce red due to ethidium bromide. Fig. 5e depicts the\nand proliferate in an uncontrollable manner to facilitate cancer devel\u00ad significant cell death brought by Ir-CN + laser treatment with the\nopment. Thus, an approach of modulating key elements in the apoptotic fluorescence of ethidium bromide, whereas the probe alone treated and\npathway can be the righteous strategy for therapy-induced tumor-cell control cells remain healthy mainly showing green fluorescence. A few\ndeath [33]. The PDT-mediated singlet oxygen generation is reported to dead cells were observed with the laser-exposed group (Ir-CN un\u00ad\nactivate some proapoptotic proteins while inhibiting some anti- treated), which was not significant as compared to Ir-CN + laser treat\u00ad\napoptotic signal cascades, ultimately leading to programmed cell ment group. Similar results were obtained with the APOP staining assay,\ndeath. As mitochondria is the powerhouse of the cell that controls redox where the dead cells take up the dye and hence be seen as pink-colored\nhomeostasis and apoptosis, mitochondria-targeting photosensitizers can cells under the bright field of microscope due to the damaged cell\nbe considered more effective candidates for photodynamic therapy [34]. membrane, while the healthy cells remain unstained (Fig. S21a).\nSince Ir-CN is found to colocalize in the mitochondria, it enables Maximum cell death was visible in the Ir-CN + laser-treated cells.\nmitochondria-mediated phototoxicity. Similar effects were manifested in trypan blue dye exclusion assay as\n The apoptotic changes are manifested by chromatin condensation, well. Trypan blue is an acidic azo dye that is impermeable to the intact\nmembrane damage, exposure of phosphatidyl serine on the outer layer cell membrane. The dye will be taken up by the dead cells with damaged\nof the plasma membrane etc. Annexin V apoptotic assay was conducted cell membranes so that the cytoplasm will be stained blue in colour [37].\nto determine the percentage of cells in each phase of cell death like early The untreated control cells with and without laser exposure and Ir-CN\napoptosis, late apoptosis, or necrosis. Here we employed Annexin V- alone treated groups mostly showed viable cells with clear cytoplasm\nFITC conjugate, which can specifically bind to the phosphatidyl serine\n\n 7\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\n\n\nFig. 5. (a) A pictorial representation of Ir-CN showing its PDT effect (b) a graph depicting the significant depletion of percentage cell viability when treated with Ir-\nCN and irradiated with laser, (c) Quantification of apoptosis by flow cytometry using Annexin V assay, and (d) graphical representation of its results (e) evaluation of\napoptotic inductions through PDT by Ir-CN in MCF-7 cells acridine orange-ethidium bromide dual staining assay (live-dead assay) (scale bar = 100 \u03bcM) (f) JC-1\nmitochondrial membrane potential assay (scale bar = 50 \u03bcM).\n\n\n\n\n 8\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\nwhile the Ir-CN treated, laser-irradiated cells showed the maximum evaluated. As depicted in the graph (Fig. S22), control, compound\npercentage of blue-colored dead cells (Fig. S21b). treated and laser-treated cells showed a normal pattern of the cell cycle\n Mitochondrial transmembrane potential (\u0394\u03a8m) can be considered as with its sub-G0 population as insignificantly low as 0.5\u20130.6%, whereas\nan essential parameter of mitochondrial function. Apoptosis is also Ir-CN treated laser irradiated cells showed not only a decrease of >20%\nknown for its characteristic feature of a significant depletion in this of the cell population in G1 phase but also a dramatic increase in those at\npotential due to enhanced membrane permeability and subsequent loss its sub-G0 phase to 27.3%. The sharp elevation in the sub-G0 cell pop\u00ad\nof electrochemical potential. The lipophilic, cationic dye JC-1 can enter ulation clearly demonstrates the happening of apoptosis induced by the\nand accumulate in the negatively charged mitochondria of healthy cells PDT effect of our compound (Fig. 6d).\nwith a normal \u0394\u03a8m. It spontaneously forms reversible complexes (J Disruption and degradation of proteins, DNA, and other bio\u00ad\naggregates) in a concentration-dependent manner with its excitation molecules that occur as a part of apoptosis show momentous changes in\nand emission in the red spectrum (maximum at ~590 nm), unlike its the Raman fingerprint patterns of the cells. Thus, Raman spectroscopy\nnaturally green fluorescent monomer. In apoptotic cells, though the dye has been successfully implemented to track the apoptotic events in the\nenters the mitochondria, JC-1 does not reach sufficient concentration to cells. The Ir-CN treated and untreated cells were darted with laser, and\ntrigger the formation of J aggregate formation occurs only to a lesser their Raman spectra were recorded. Apoptotic behavior was expected\ndegree due to a lesser negative environment, thus giving green fluo\u00ad only in the former group of cells. A decline in intensity of the peak at\nrescence of the monomer. The reduction in the red-to-green fluorescence 838 cm\u2212 1 which corresponds to the breakage of the phosphodiester\nintensity ratio therefore indicates mitochondrial depolarization [38]. backbone of DNA resulting in the purine base exposure, was observed in\nSince Ir-CN also exhibits red fluorescence, the experiment was carried the cells compound treated-laser shined cells, as compared to the other\nout 24 h later for Ir-CN-containing wells, to allow the complex to have group. An increase in the peak intensity of the former sample at 1585\nsufficient time to undergo metabolism, so that it does not interfere with cm\u2212 1 could be assigned to the N7-H vibration of guanine and adenine\nthe fluorescence of the JC-1 aggregate. As indicated in Fig. 5f the suf\u00ad due to their exposure, clearly to DNA fragmentation. The C-H defor\u00ad\nficient entry of the dye and thereby the J aggregate formation is found to mation of these bases are also evident from the heightening of the peak\nbe difficult in case of cells treated with Ir-CN and irradiated with laser, at 1400\u20131480 cm\u2212 1. The disruption of protein tertiary structure and\ncompared to others. The low aggregate: monomer ratio in such cells denaturation becomes apparent through the sharp hike observed in the\nproves the reduction in the MMP achieved as a consequence of PDT- peak at 1000 cm\u2212 1 in the former group of cells which is obviously\ninduced apoptosis by Ir-CN (Fig. S27). suggestive of the exposure of hydrophobic amino acids such as\n Yet another hallmark of apoptotic cells, the phenomenon of nuclear phenylalanine, tryptophan and tyrosine due to the absence of secured\ncondensation was also observable in the Ir-CN+ laser-treated cells with positioning and protection within the intricately folded proteins [42,43]\nthe staining of nuclear dye Hoechst 33342 (Fig. 6c) as compared to other (Fig. 6e).\ntreatment groups. Here, the nuclei of healthy cells will appear in a round In the realm of apoptosis, Cytochrome c (Cyt c) unquestionably plays\nshape with uniform staining whereas the apoptotic cell will have a leading role. It is resident of the mitochondrial intermembrane space\nbrighter and irregularly shaped nuclei. Together with this, inter\u00ad and normally functions in the electron shuttle between complex III and\nnucleosomal degradation of double-stranded DNA by the action of IV. Cyt c is discharged into the cytosol as a result of apoptotic stimuli,\ncaspase-activated DNase (CAD) on the genomic DNA is also an observ\u00ad where, it facilitates allosteric activation and formation of a hepta-\nable change associated with apoptosis. This leads to fragments of sizes in oligomer with the adapter molecule Apaf-1 (apoptosis-protease acti\u00ad\nmultiples of 180-200 bp, which when separated and visualized on an vating factor-1), leading to apoptosome formation [44]. Here we have\nagarose gel electrophoresis, form a distinctive ladder pattern [39]. As in recorded the SERS spectra to track the spectral signature of cytochrome\nFig. 6a, the typical laddering pattern of DNA fragments on the gel was c which has been released in the initial stage of apoptosis. The Raman\nobserved in case of the Ir-CN treated, laser-irradiated sample, compared peaks characteristic of cytochrome c at 750 cm\u2212 1 and 1050 cm\u2212 1 [42]\nto the others. This also points towards the apoptosis-inducing effect of Ir- along with the other peaks corresponding to apoptosis were observed\nCN\u2019s PDT. only from Ir-CN treated cells, after one and two hours of laser irradiation\n The SERS spectra of isolated DNA from each sample were recorded (Fig. 6f).\nfor the reconfirmation of the characteristic DNA fragmentation associ\u00ad Further, to confirm the ROS generation that happened upon laser\nated with apoptosis. As shown in Fig. 6b, several signature Raman peaks irradiation on Ir-CN treated cells, DCFDA staining was performed fol\u00ad\nof DNA were obtained of which some show a significant shift or decrease lowed by flow cytometric analysis. As expected, the maximum ROS\nin intensity in the case of DNA from the Ir-CN treated, laser-irradiated generation was found in the Ir-CN treated laser irradiated group as\nsamples. The intensities of peaks at 766 cm\u2212 1, 782 cm\u2212 1 and 877 compared to the others (fig). By virtue of the PDT effect, disruption of\ncm\u2212 1 corresponding to the pyrimidine, DNA mode vibration and C-O-C respiratory chain and thereby a decline in the ATP level was also ex\u00ad\nvibration of sugar backbone respectively were found to be decreasing pected. To substantiate this claim, ATP determination assay was per\u00ad\nand disappearing in the case of Ir-CN treated, laser irradiated sample. formed to estimate the amount of ATP generated in each group. The\nSimilar was the case of peaks at 1002 cm\u2212 1 (C-C aromatic ring vibration percentage of ATP was observed to fall below 25% in the case of the Ir-\nof N bases), 1085\u20131098 cm\u2212 1 phosphate backbone stretching, 1175 CN treated, laser-irradiated group as compared to the untreated control\ncm\u2212 1 Cytosine and Guanine,1304 cm\u2212 1 for Adenine and Cytosine, (Fig. 6h). This decrease in ATP level was found to be significantly\n1455\u2212 1460 & 1575 cm\u2212 1 for DNA bases ring breathing, nucleic acid different from control + laser treated as well as Ir-CN treated groups\nmode and 1605 cm\u2212 1 Cytosine peaks. Whereas, 1620\u20131750 cm\u2212 1 peak (***p < 0.001) that further authenticate the claim.\nfor base pairing and H bond between N bases showed remarkable peak\nenhancement which may be due to the DNA Breakage and exposure of 3. Conclusions\nthe double helical structure to the SERS substrate (Table S5).\n Cell cycle arrest (CCA) is one of the prime candidates for effective In conclusion, our investigation focused on evaluating the photo\u00ad\ncancer therapy and a characteristic of various therapeutic modalities therapeutic potential of two cyclometalated Iridium (III) molecular\n[40]. Though the mechanism remains unclear, PDT is also reported to probes, Ir-CN and Ir-H, with a specific emphasis on mitochondria-\nhave a biological effect on CCA in cancer cells [41]. To accurately mediated cell death. Through a comprehensive analysis, Ir-CN\nidentify the phase of cell cycle being arrested as a part of the PDT by Ir- emerged as the preferred candidate for further investigation owing to\nCN, a propidium iodide-based, flow cytometric analysis of the DNA its superior photosensitizing characteristics and a more favorable\ncontent in the cells was done. Based on the DNA content in them, the toxicity profile compared to Ir-H. The notable features of Ir-CN,\npercentage of cells in each of the phases (sub-G0, G1, S and G2) were including a substantial singlet oxygen quantum yield, were identified\n\n 9\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\n\n\nFig. 6. Nuclear fragmentation and condensation induced by Ir-CN upon laser treatment proven with (a) DNA laddering by AGE (b) SERS analysis of DNA frag\u00ad\nmentation (A- control, B- control+laser, C- Ir-CN, D- Ir-CN + laser), and (c) Hoescht nuclear staining showing heterochromatinization (scale bar = 100 \u03bcM) (d) Cell\ncycle analysis clearly showing G1 phase arrest (e) SERS spectra showing the peaks characteristic of apoptosis in Ir-CN treated laser darted cells (f) SERS spectra\nrevealing Cyt C release occurred in the initial stages of apoptosis induced by the PDT effect of Ir-CN (g) ROS generation study using flow cytometric analysis\n\n\n\n\n 10\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\nemploying DCFDA staining method (h) Estimation of respiratory chain damage ATP determination assay (control vs control+laser- **P < 0.01, Ir-CN vs Ir-CN + laser\n*** P < 0.001, control+laser vs Ir-CN + laser ***P < 0.001).\n\n\nas pivotal attributes for its potential application as a photodynamic trypsinization, after rinsing with PBS (Phosphate Buffered Saline, pH\ntherapy (PDT) agent. The robust spin-orbit coupling associated with the 7.4). Trypsin-EDTA solution 10\u00d7 (0.5 Trypsin, 0.2% EDTA in 0.85%\ncentral iridium metal played a key role in facilitating efficient singlet normal saline, Gibco) was used for detaching cells from the culture flask.\noxygen generation, enhancing the overall efficacy of Ir-CN in PDT. The All the cell images were captured using Nikon Eclipse TS100 with Nikon\npronounced red fluorescence emitted by Ir-CN not only allowed for NIS Elements software unless mentioned. All OD measurements for\neffective visualization of its cellular uptake but also contributed to its biological studies were done using Synergy H1 microplate reader, Bio\ntargeted localization within mitochondria. The positive charge carried Tek Instruments, USA. All the flow cytometry and its data analysis were\nby the central metal atom in Ir-CN was instrumental in enhancing its done using BD FACSAria\u2122 II and BD FACSDiva\u2122 software respectively.\naffinity for mitochondria, enabling specific PDT effects within these The DNA is extracted from the cells using the genomic DNA extrac\u00ad\ncellular organelles. Experimental evidence supporting the induction of tion kit (Geneaid Blood/Cell DNA Mini Kit (GB100) and the purity of the\napoptosis by Ir-CN in MCF-7 cells was robust, as demonstrated through DNA is estimated using nanodrop (Synergy H1, Biotek). The samples are\nAnnexin V-FITC assay, revealing a predominant apoptotic effect with a made to undergo agarose gel electrophoresis (0.8% agarose). The lad\u00ad\nsignificant proportion of cells in the early apoptotic phase. This obser\u00ad dering pattern on the gel is photographed using ChemiDoc imaging\nvation was further corroborated by monitoring the release of system (Bio-rad).\nCytochrome-C using Surface-Enhanced Raman Spectroscopy (SERS). The following experiments were performed. The detailed method\u00ad\nCharacteristics indicative of Ir-CN PDT-induced apoptosis, such as cell ology of each experiment is provided in the supporting information (SI).\nnuclear and membrane damage, DNA condensation, and protein\ndegradation, were observed through fluorescence assays and SERS 4.1. Design and Synthesis of the Cyclometalated Ir(III) Complexes\nmeasurements. The DCFDA assay as well as the cellular ATP estimation\nconfirmed the enhanced ROS generation and declined ATP levels due to The detailed steps of synthesis for the as-developed candidate\nIr-CN PDT. Thus, the mechanism of apoptotic induction is supposed to cyclometalated iridium (III) complexes Ir-CN and Ir-H are given in the\nbe mediated via ROS-induced disruption of the electron transport chain supporting information. The prepared complexes were well character\u00ad\ncausing energy deprivation in the cells. These findings collectively un\u00ad ized through 1H NMR, 13C NMR, and HRMS spectroscopic techniques.\nderscore the potential of Ir-CN as a PDT molecular probe with promising\ntherapeutic benefits. The outcomes of this study pave the way for future 4.2. Photophysical and Stability Assessment of Ir-CN and Ir-H\nclinical translation, offering a novel avenue for the development of\ntargeted and effective phototherapeutic strategies in the realm of cancer The absorbance, emission, and Raman spectra of the compounds\ntreatment. were recorded. The compound was exposed to long UV at 405 nm laser\n in order to assess the maximum duration of photostability. pH titration\n4. Materials and Methods at several pH levels ranging from 4.5 to 9.5 was utilized to confirm the\n stability of compounds\u2019 absorption and emission characteristics in this\n The chemicals including 4,4\u2032-dibromo-2,2\u2032-bipyridine, 4-cyano\u00ad range of pH environments.\nphenyl boronic acid, phenylboronic acid, potassium carbonate, Pd\n(PPh3)4, IrCl3\u22c5xH2O, 2-phenyl pyridine, 2-methoxy ethanol, methyl io\u00ad 4.3. Photooxidation and Singlet Oxygen Generation Studies of Ir-CN and\ndide, diphenylisobenzofuran were purchased from TCI Chemicals and Ir-H\nTBAB (Tetrabutylammonium bromide) was procured from Sigma\nAldrich. All solvents were purchased from Merck and were of reagent Using the well-known 1O2 scavenger dye 1,3-diphenylisobenzofuran\ngrade. Standard procedures were followed for drying and distilling all (DPBF) (Fig. 2c) and a structurally significant molecule [Ru(Bpy)3]Cl2 as\nsolvents prior to use. All NMR spectra were recorded on a Bruker 500 standard reference material, the Ir-CN was examined for its ability to\nMHz FT-NMR (model) spectrometer at 25 \u25e6 C. The chemical shift (\u03b4) data generate 1O2. The singlet oxygen production in the solution state was\nand coupling constant (J) values were given in parts per million (ppm) further confirmed by the well-known singlet oxygen indicator Singlet\nand Hertz (Hz), respectively. High-resolution mass spectra (HRMS) were Oxygen Sensor Green (SOSG).\nrecorded on a Thermo Scientific Exactive ESI-MS spectrophotometer.\nUV/Visible spectra were obtained by using a Shimadzu UV-2600 UV\u2013Vis\n 4.4. Evaluation of Cytotoxicity and Cellular Internalization of Ir-CN and\nspectrophotometer and emission spectra were recorded on a Fluoromax-\n Ir-H\n4 Spectrofluorometer with a 1 cm quartz cuvette. All experiments were\ncarried out at 298 K unless otherwise specified. SERS experiments were\n MTT assay was used to determine the extent of dark toxicity rendered\nperformed using a WITec Raman microscope (WITec, Inc., Germany)\n by the complexes. Based on the results of the MTT assay, the better\ncontaining 600 g/mm grating and Peltier cooled charge-coupled device\n candidate was chosen and its time and concentration-dependent inter\u00ad\ndetector unit. The samples were excited using a 633 nm laser with 10\n nalization studies were conducted.\nmW power, and the Stoke-shifted Raman spectra were collected in the\nregion of 300\u20132000 cm\u2212 1 with a resolution of 1 cm\u2212 1 and an integration\n 4.5. Assessment of Mitochondrial Targetability of Ir-CN\ntime of 1 s and 10 accumulations. Prior to each measurement, calibra\u00ad\ntion was done with a silicon standard (Raman peak at 520 cm\u2212 1). WITec\n Even though it was hypothesized that by virtue of the positive charge\nProject Plus (v 5.2) software package was employed for data processing.\n of the complex would specifically move to the mitochondria, colocali\u00ad\n In vitro, phototherapeutic potential, and all other imaging studies of\n zation studies using mito, lyso, and nuclear trackers were conducted to\nIr-CN were performed using MCF-7 cell line procured from ATCC. The\n specifically identify the organelle to which the complex is localized\ncells were maintained in tissue culture flasks (T25 and T75) with Dul\u00ad\n within the cell.\nbecco\u2019s Modified Eagle Medium (DMEM, Sigma Aldrich) supplemented\nwith 10% Fetal Bovine Serum (FBS, HiMedia), 1% antibiotic antimycotic\nsolution (Anti-Anti 100\u00d7, Gibco). Flasks were incubated at 37 \u25e6 C and 5% 4.6. Assessment of the PDT Effects of Ir-CN on MCF-7 Cells\nCO2. The cells were sub-cultured upon attaining 80% confluency by\n MTT assay was performed on Ir-CN treated, laser (405 nm) irradiated\n\n 11\n\fC. Reghukumar et al. Journal of Photochemistry & Photobiology, B: Biology 250 (2024) 112832\n\n\ncells to estimate the cytotoxicity caused upon treatment. The generation References\nefficiency of reactive oxygen species as a part of the PDT effect of the\ncompound was also confirmed by the DCFDA assay by fluorescent im\u00ad [1] J. Rak, Cancer: organ-seeking vesicles, Nature. 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Guo, Recent advances in noble metal complex\nreview & editing, Validation, Software, Formal analysis, Data curation. based photodynamic therapy, Chem. Sci. 13 (2022) 5085\u20135106, https://doi.org/\n 10.1039/D1SC05478C.\nVishnu Priya Murali: Writing \u2013 review & editing, Visualization, Vali\u00ad\n [14] S. Banerjee, A.R. Chakravarty, Metal complexes of curcumin for cellular imaging,\ndation, Software, Formal analysis, Data curation. Pilankatta K. Ramya: targeting, and photoinduced anticancer activity, Acc. Chem. Res. 48 (2015)\nSoftware, Data curation. Kokkuvayil Vasu Radhakrishnan: Supervi\u00ad 2075\u20132083, https://doi.org/10.1021/acs.accounts.5b00127.\nsion, Funding acquisition. Kaustabh Kumar Maiti: Writing \u2013 review & [15] O.J. Stacey, S.J.A. Pope, New avenues in the design and potential application of\n metal complexes for photodynamic therapy, RSC Adv. 3 (2013) 25550\u201325564,\nediting, Supervision, Resources, Project administration, Funding https://doi.org/10.1039/c3ra45219k.\nacquisition, Conceptualization. [16] S. Shamjith, M.M. Joseph, V.P. Murali, G.S. Remya, J.B. Nair, C.H. Suresh, K.\n K. Maiti, NADH-depletion triggered energy shutting with cyclometalated iridium\n (III) complex enabled bimodal luminescence-SERS sensing and photodynamic\n therapy, Biosens. Bioelectron. 204 (2022), https://doi.org/10.1016/j.\nDeclaration of Competing Interest bios.2022.114087.\n [17] X. Cai, K.N. Wang, W. Ma, Y. Yang, G. Chen, H. Fu, C. Cui, Z. Yu, X. 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