Novel tris-bipyridine based Ru(II) complexes as type-I/-II photosensitizers for antitumor photodynamic therapy through ferroptosis and immunogenic cell death.

{"full_text": " European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nNovel tris-bipyridine based Ru(II) complexes as type-I/-II photosensitizers\nfor antitumor photodynamic therapy through ferroptosis and immunogenic\ncell death\nHongwei Zheng a,b,1 , Kai Wang b,1 , Dongliang Ji a,b,1 , Xiao Liu a,b, Chen Wang a,b ,\nYangyang Jiang b, Zihan Jia b , Biao Xiong b,***, Yong Ling a,b,**, Jiefei Miao a,b,*\na\n Department of Oncology, Department of Pharmacy, Affiliated Hospital of Nantong University, Nantong 226001, China\nb\n School of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong Key Laboratory of Small Molecular Drug Innovation,\nNantong University, Nantong 226001, China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nHandling Editor: Dr. Z Liu Ru(II) complexes have attracted attention as photosensitizers for their promising photodynamic properties.\n Herein, novel tris-bipyridine based Ru(II) complexes (6a-e) were synthesized by introducing saturated hetero-\nKeywords: cycles to improve photodynamic properties and lipid-water partition coefficients. Among them, 6d demonstrated\nRu(II) complexes significant phototoxicity towards three cancer cells, with IC50 values of 5.66\u20137.17 \u03bcM, exceeding values in dark\nTris-bipyridine\n (IC50s > 100 \u03bcM). Under hypoxic conditions, 6d maintained excellent photodynamic activity in A549 cells, with\nPhotodynamic therapy (PDT)\n PI values exceeding 24, highlighting its potential for highly effective type-I/-II photodynamic therapy by\nAntitumor activity\nFerroptosis inducing ROS generation, oxidative stress, and mitochondrial damage. Additionally, it induced ferroptosis and\n immunogenic cell death of A549 cells by regulating the expression of relevant markers. Finally, 6d remarkably\n inhibited the growth of A549 transplanted tumor growth by 95.4 %. This Ru(II) complex shows great potential\n for cancer treatment with its potent photodynamic activity and diverse mechanisms of tumor cell death.\n\n\n\n\n1. Introduction responses. PDT offers distinct advantages over traditional methods,\n including precise spatio-temporal control, non-resistance, and minimal\n The ongoing battle against cancer remains a paramount concern in invasiveness [10]. In cases where surgery is not an option or other\nglobal public health, marked by its persistently high morbidity and treatments have failed or been declined, PDT can serve as an option or as\nmortality rates [1\u20133]. Among the array of malignancies, lung cancer part of combination therapy.\nstands out as particularly daunting, often diagnosed at advanced stages Three essential non-toxic components are involved in the process of\nwhere conventional treatments like surgery and radiation become PDT: the photosensitizers (PSs), laser light of specific wavelengths, and\nineffective [4,5]. Furthermore, its intrinsic or acquired multidrug oxygen [11]. Through photochemical reactions, PSs generate toxic ROS\nresistance complicates therapeutic interventions, necessitating explo- to destroy tumor cells. Notably, while type II photochemical reactions\nration of alternative modalities [6,7]. (which generate 1O2) dominate current research, type I reactions (which\n In recent decades, photodynamic therapy (PDT) has emerged as a generate \u2022O\u22122 and \u2022OH, etc.) are less dependent on oxygen and hold\npromising avenue for the treatment of certain forms of tumors, including more promise for enhanced therapeutic outcomes in hypoxic tumor\nlung cancer, colorectal cancer (CRC), melanoma, and so on [8,9]. This microenvironments [12\u201314]. Therefore, it is of great significance to\nmethod leverages reactive oxygen species (ROS) generated upon light explore type I/II photosensitizers, which can overcome tumor hypoxia.\nactivation to induce apoptosis, necrosis, vascular damage, and immune The quest for optimal PSs has indeed been a long-standing endeavor,\n\n\n * Corresponding author. Department of Oncology, Department of Pharmacy, Affiliated Hospital of Nantong University, Nantong 226001, China.\n ** Corresponding author. Department of Oncology, Department of Pharmacy, Affiliated Hospital of Nantong University, Nantong, 226001, China.\n *** Corresponding author. School of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong Key Laboratory of\nSmall Molecular Drug Innovation, Nantong University, Nantong 226001, China.\n E-mail addresses: hsiung1987@ntu.edu.cn (B. Xiong), LYYY111@sina.com (Y. Ling), miaojfntu@163.com (J. Miao).\n 1\n These authors contributed equally.\n\nhttps://doi.org/10.1016/j.ejmech.2024.116909\nReceived 29 June 2024; Received in revised form 15 September 2024; Accepted 23 September 2024\nAvailable online 24 September 2024\n0223-5234/\u00a9 2024 Elsevier Masson SAS. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n\n\n Fig. 1. The chemical structure of four clinical Ru complexes: NAMI-A, KP1019, KP1339, and TLD1443.\n\n\n\ntracing back to the pioneering breakthrough of haematoporphyrin de- Phase IB clinical trials for PDT in bladder carcinoma treatment, under-\nrivative (HpD) in the 1960s [15,16]. The tetrapyrrole-based photosen- scoring the translational potential of Ru(II) complexes [30,31]. There-\nsitizers, such as porphyrins, chlorins, and phthalocyanines, stand out as fore, the development of Ru(II) complexes for PDT holds significant\nthe most extensively researched class of photosensitizers, with certain clinical value in cancer therapy.\nmembers already receiving clinical approval [17,18]. However, because Given that bipyridine is an important bidentate ligand with good\nof the similar structure core, they often suffer from inherent limitations planarity and high designability, its molecular optical and physical\nrelated to solubility, photostability, pharmacokinetics, and so on properties can be optimized by structural modification. Researches have\n[19\u201321]. In contrast, metal-based drugs, particularly certain Ru com- shown that complexes formed with polypyridine ligands have the ability\nplexes, present an extensive array of biological applications, with sig- to emit fluorescence persistently, display remarkable large Stokes shifts,\nnificant promise in PDT [22,23]. Unlike the flat quadrilateral showcase notable single and two-photon absorption abilities, and up-\narrangement in cisplatin, Ru complexes display a d6 hexa-coordinated hold fluorescence stability under physiological conditions [32\u201334].\noctahedral structure with a 3D arrangement of ligands, considerably These combined features bestow upon Ru(II) polypyridine complexes\nwidening the possibilities for modification [24]. The reasonable choice the potential for effective photosensitization, a crucial mechanism in\nof ligands can endow Ru complexes with a wide range of superior PDT. Herein, we employed 4,4\u2032-dimethyl-2,2\u2032-bipyridine as the bidentate\nproperties, encompassing the improvement of solubility, photodynamic ligand parent nucleus and introduced saturated heterocycles to improve\ncharacteristics, ROS yield, and so on [25,26]. Several Ru complexes have the lipid-water partition coefficients (log P) and photodynamic activ-\nentered clinical research and shown potential for tumor therapy (Fig. 1). ities, resulting in the novel bidentate nitrogen ligand, bipyridine de-\nNAMI-A and KP1019 have been excluded from clinical use due to their rivatives 5a-e. Finally, they were direct coordinated to the Ru metal\npoor anti-tumor effects, limited water solubility, and severe side effects center to obtain functional tris-bipyridine Ru(II) complexes 6a-e,\n[27,28]. To address the low water solubility of KP1019, researchers enabling facile large-scale synthesis (Fig. 2). It is expected to enhance\nhave developed a more soluble sodium salt complex, KP1339, which is their photodynamic activities while also improving log P values to\ncurrently undergoing Phase 1/2 clinical trials for the treatment of promote cellular absorption. Moreover, their potential inhibitory and/or\nvarious types of tumors [29]. The mentioned three complexes are uti- damaging effects on tumor cells through PDT, along with preliminary\nlized as chemotherapy agents. Notably, the inert Ru(II) polypyridyl mechanisms such as cell ferroptosis and immunogenic cell death, were\ncomplex TLD1433, pioneered by McFarland et al., has advanced to further explored.\n\n\n\n\n Fig. 2. The chemical structure of novel Ru(II) complexes 6a-e and the multifaceted mechanisms.\n\n\n 2\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n on, while the type II pathway (energy transfer process) yields 1O2. The\n majority of reported PSs are based on type II mechanism, which is\n greatly relying on oxygen [11]. Dihydroethidium (DHE), 4-hydroxyphe-\n nyl-fluorescein (HPF), and 1,3-Diphenylisobenzofuran (DPBF) were\n utilized to further probe the ability of the five complexes to generate\n \u2022O\u22122 , \u2022OH, and 1O2, aiming to identify their specific photodynamic\n pathways (Fig. 3d\u2212 f and S2\u2212 4). The fluorescence intensity of DHE and\n HPF exhibited varying increases upon irradiation, reflecting the\n different \u2022O\u22122 and \u2022OH generation capability of 6a-e. As an indirect\n method, the absorbance of DPBF at 415 nm significantly decreased after\n incubation with 6a-e followed by exposure to 520 nm light, and the 1O2\n quantum yields (\u03a6\u0394) of 6a-e was subsequently calculated using [Ru\n (bpy)3](PF6)2 (RuB) as a reference [35]. As illustrated in Table 1, the\n results indicate that the 1O2 generation capability of 6a-e follows this\n order: 6a (\u03a6\u0394 = 0.83) > 6d (\u03a6\u0394 = 0.81) > 6e (\u03a6\u0394 = 0.77) > RuB (\u03a6\u0394 =\n 0.73) > 6c (\u03a6\u0394 = 0.57) > 6b (\u03a6\u0394 = 0.23).\n Combining the subsequent phototoxicity data (Table 2), complexes\n 6d and 6a were chosen for the subsequent studies. In the context of\nScheme 1. Synthetic scheme for Ru(II) complexes 6a-ea photosensitizers, good photostability is deemed to be a crucial prereq-\na\n (a) SeO2, 1,4-dioxane, 100 \u25e6 C, 24 h, 40 %; (b) NaBH4, MeOH, r. t., 2 h, 88 %; uisite. Therefore, the photostability of 6d and 6a was assessed upon\n(c) HBr, H2SO4, 110 \u25e6 C, 12 h, 85 %; (d) diverse saturated heterocycles, K2CO3,\n continuous irradiation (520 nm, 200 mW/cm2) in methanol and moni-\nMeCN, 85 \u25e6 C, 12 h, 71\u201383 %; (e) RuCl3\u22c5H2O, (CH2OH)2, 120 \u25e6 C, 6 h, and then\n toring of the potential conversion of the complexes by UV\u2013visible ab-\nNH4PF6, three steaming water, r. t., 10 min, 51\u201357 %.\n sorption spectra. Promisingly, no significantly decrease was observed,\n indicative of the high photostability of them (Fig. S5). Moreover, the\n2. Results and discussion\n relative fluorescence quantum yields of 6a and 6d were assessed in a\n mixed solution of methanol and ethanol (1:4, v/v) using RuB as the\n2.1. Synthesis and characterization\n reference (\u03a6F = 0.328) [36]. 6d demonstrated a higher fluorescence\n quantum yield of 0.329, surpassing that of 6a which stands at 0.273.\n Herein, 4,4\u2032-dimethyl-2,2\u2032-bipyridine was selected as the parent nu-\n Electron spin resonance (ESR) spectroscopy was employed to further\ncleus for the bidentate ligand, and saturated heterocycles were intro-\n confirmed the \u2022O2\u2212 and 1O2 generation ability of 6d. 5,5-dimethyl-1-\nduced to enhance the log P. The synthesis of Ru(II) complexes 6a-e is\n pyrroline N-oxide (DMPO) and 2,2,6,6-tetramethyl-4-piperidone\ndescribed in Scheme 1. Starting with 4,4\u2032-dimethyl-2,2\u2032-bipyridine (1), it\n (TEMP) were employed as the \u2022O\u22122 and 1O2 trapping agents, respec-\nwas oxidized using selenium dioxide (SeO2) to yield compound 2.\n tively. Clearly, a strong characteristic signal of \u2022O\u22122 was detected in the\nSubsequent reduction with sodium borohydride (NaBH4) afforded\n methanol solution of 6d under 520 nm illumination. No apparent \u2022O\u22122\ncompound 3. After bromination with phosphorus tribromide (PBr3), the\n signal was observed in the absence of light (Fig. 3g). These results are\nresulting compound 4 was obtained. Next, various nitrogen heterocycles\n consistent with the DHE fluorescence attenuation curves, confirming\nwere introduced to compound 4 to obtain novel bidentate nitrogen li-\n that \u2022O\u22122 was generated only when 6d was under photoexcitation\ngands 5a-e. These generated ligands were then coordinated to the metal\n (Fig. 3d). Interesting, complex 6d exhibited notably reduced \u2022OH pro-\ncenter of RuCl3, and ammonium hexafluorophosphate (NH4PF6) was\n duction compared to \u2022O\u22122 , implying its reliance primarily on \u2022O\u22122 to\nadded to yield the corresponding salts, leading to the formation of Ru(II)\n mediate type I PDT (Fig. 3e and h). The characteristic triplet ESR signal\ncomplexes 6a-e. The synthesized compounds were characterized using\n1 of 1O2 was also observed after 520 nm irradiation (Fig. 3i). Taken\n H and 13C NMR spectroscopy, and ESI-HRMS as described in the Sup-\n together, these findings imply that further mechanism investigation into\nporting Information (Fig. S11\u2212 40).\n photodynamic activity of 6d was worthwhile due to its significant po-\n tential as a potent generator of ROS through both type I and type II\n2.2. Photophysical and photodynamic properties processes.\n\n The UV\u2013visible absorption and fluorescence spectra of Ru(II) com- 2.3. In vitro phototoxicity\nplexes 6a-e were recorded at room temperature, using methanol as the\nsolvent, to investigate their photophysical characteristics. All five We conducted further toxicity evaluations of 6a-e under both dark\ncomplexes exhibited a comparable profile, indicating that the available condition and irradiation against various cell lines, including HT29\nelectronic transitions, as well as the ground and excited states within the human colon cancer cells, A549 human lung cancer cells, and 4T1\ncomplexes, were qualitatively alike (Fig. 3a). They displayed broad mouse breast cancer cells. The cell inhibition rates of all complexes at\npeaks spanning the wavelength range of 400\u2013500 nm (with maxima at varying concentrations are depicted in Fig. 4 and S6-7, while the\napproximately 459 nm) ascribed to the Metal-to-Ligand Charge Transfer resulting IC50 values and photocytotoxicity indices (PI) of 6a-e are\n(MLCT) from Ru (d\u03c0) to the bpy ligand (\u03c0*) transitions. Moreover, all calculated and presented in Table 2. It is evident that among all com-\ncomplexes except for 6b displayed a high molar extinction coefficient plexes, the dark toxicity of 6d to these cell lines was minimal in both\n(>104 M\u2212 1 cm\u2212 1), rendering them highly attractive as photosensitizers under normoxic and hypoxic conditions (IC50s > 100 \u03bcM) (Table S1).\nfor PDT (Table 1). Under ambient conditions, 6a-e displayed red emis- However, under 520 nm photoirradiation, the photocytotoxicity of 6d\nsion with a peak wavelength approximately 622 nm upon excitation at increased significantly (IC50s = 4.16\u20139.12 \u03bcM), with PI all exceeding\n459 nm (Fig. 3b). 10.9. Interestingly, 6d demonstrated higher photocytotoxicity against\n Firstly, the generation of total ROS by 6a-e was assessed using 2\u2032,7\u2032- HT29 and A549 cells in hypoxia, indicating its ability to overcome the\ndichlorofluorescin (DCFH, ROS probe), the fluorescence attenuation limitation of O2 concentration through type I and type II photodynamic\ncurves indicated that all five complexes could generate a significant reactions. These experimental results further validate our previous ex-\namount of ROS upon irradiation (Fig. 3c and S1). PDT can trigger cancer periments and attest to the potential of complex 6d to serve as a type-I/-\ncell apoptosis via two distinct pathways based on the ROS generated: the II photosensitizer.\ntype I pathway (electron transfer process) produces \u2022O\u22122 , \u2022OH, and so The partition coefficient between lipid and water affects the\n\n 3\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n\n\nFig. 3. Photophysical and photodynamic properties of Ru complexes 6a-e. UV\u2013Vis absorption (a) and fluorescence (b) spectra of 6a-e (10 \u03bcM) measured in methanol\nsolution at room temperature, \u03bbex = 460 nm. (c) The fluorescence changes of 6d (10 \u03bcM) in methanol solution containing DCFH (total ROS probe) under laser\nirradiation. (d) The fluorescence changes of 6d (10 \u03bcM) in methanol solution containing DHE (\u2022O\u22122 probe) under laser irradiation. (e) The fluorescence changes of 6d\n(10 \u03bcM) in methanol solution containing HPF (\u2022OH probe) under laser irradiation. (f) The absorption attenuation curve of DPBF measured in methanol solution of 6d\nunder laser irradiation. EPR signals for the \u2022O\u22122 (g), \u2022OH (h), and 1O2 (i) characterization of 6d upon irradiation or in the dark. Light dose: 520 nm, 200 mW/cm2.\n\n\nTable 1\nThe log P values measured by the classical shake-flask method and photophysical properties of complexes 6a\u2013e.\n Compd. \u03bbabs (nm) \u03b5 (M\u2212 1 cm\u2212 1) \u03bbem (nm) stokes shift (nm) \u03a6\u0394 \u03a6F log P\n\n RuB 456 16,200 609 153 0.73 0.328 \u2212 1.91 \u00b1 0.21\n 6a 459 12,607 623 164 0.83 0.273 0.35 \u00b1 0.02\n 6b 459 8085 618 159 0.23 NCa \u2212 2.51 \u00b1 0.01\n 6c 459 12,460 618 159 0.57 NC \u2212 1.33 \u00b1 0.05\n 6d 459 12,814 622 163 0.81 0.329 0.04 \u00b1 0.02\n 6e 459 16,027 617 158 0.77 NC 0.50 \u00b1 0.07\n a\n NC: not calculated.\n\n\n\n\ndistribution, efficacy, and safety of drugs in the body. Improving the 5.8 times that of the positive control RuB (Figs. S8c and d). Combining\npartition coefficient could potentially enhance cellular uptake and in- with the log P of the three complexes, 6d maintained a moderate value,\ncrease the effectiveness of anticancer drugs. The log P of complexes 6a-e which may account for its favorable cellular uptake behavior.\nhave been calculated and listed in Table 1, with values of 0.35, \u2212 2.51, The selective phototoxic effect of 6d against A549 cells was also\n\u2212 1.33, 0.04, and 0.50, four of which were higher than RuB (log P = further visually evaluated by the live and dead cell staining with calcein-\n\u2212 1.91), indicating easier cellular entry. Inspired by the excellent AM and propidium iodide (PI) dyes. Obviously, only irradiation or 6d\nphotodynamic properties and phototoxicity of complexes 6a, 6d, and itself failed to cause a certain amount of cell death. However, 6d upon\n6e, flow cytometry was employed to evaluate their uptake by A549 irradiation could induce cell death efficiently, indicated by the appear-\ntumor cells. Initially, we investigated the cellular internalization of 6d at ance of red fluorescence from PI (Fig. 5c). Similar experimental results\nvarious time points and observed maximal uptake at 2 h, which then were observed in the 6a-treated 4T1 cells (Fig. 5a). Notably, 6d group\nslightly decreased by 4 h (Figs. S8a and b). Subsequently, comparison of exhibited a substantial reduction in the red/green fluorescence ratio\nthe internalization of the three complexes at 2 h revealed that 6d from 7.67 to 0.89 between the dark and light conditions, whereas the 6a\nexhibited significantly more and faster cellular uptake, approximately group decreased from 12.08 to 0.32 (Fig. 5b and d).\n\n\n 4\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\nTable 2\nIC50 values in the dark or under irradiation of complexes 6a-e toward three cancer cell lines.\n Compd. Conditions In vitro antiproliferative activity (IC50, \u03bcM)\n\n HT29 A549 4T1\n\n RuB normoxiaa Dark >100 >100 >100\n Light 14.90 \u00b1 1.70 27.90 \u00b1 1.30 95.62 \u00b1 16.16\n PId >6.71 >3.58 >1.05\n 6a normoxia Dark >100 >100 84.53 \u00b1 8.88\n Light 8.01 \u00b1 0.67 9.39 \u00b1 0.45 11.22 \u00b1 0.36\n PI > 12.48 > 10.65 7.53\n 6b normoxia Dark >100 >100 69.59 \u00b1 5.41\n Light 27.30 \u00b1 2.46 22.61 \u00b1 1.48 20.06 \u00b1 0.10\n PI >3.66 >4.42 3.47\n 6c normoxia Dark >100 >100 >100\n Light >100 >100 >100\n PI NCc NC NC\n 6d normoxia Dark >100 >100 >100\n Light 5.78 \u00b1 1.86 5.66 \u00b1 0.53 7.17 \u00b1 0.51\n PI > 17.30 > 17.67 > 13.95\n 6d hypoxiab Dark >100 >100 >100\n Light 5.09 \u00b1 0.41 4.16 \u00b1 0.18 9.12 \u00b1 0.25\n PI > 19.65 > 24.04 > 10.96\n 6e normoxia Dark >100 >100 >100\n Light 5.88 \u00b1 0.21 7.90 \u00b1 0.04 9.99 \u00b1 1.59\n PI > 17.00 > 12.66 > 10.01\n a\n Cytotoxicity of the tested compound incubated for 24 h in normoxia (21 % O2).\n b\n Cytotoxicity of the tested compound incubated for 24 h in hypoxia (1 % O2).\n c\n NC: not calculated.\n d\n PI: phototoxic indices, IC50, dark/IC50, irradiation. Irradiation: 520 nm, 200 mW/cm2, 10 min.\n\n\n\n\nFig. 4. Cytotoxicity and phototoxicity of RuB (a), 6a (b), 6b (c), 6c (d), 6d (e), and 6e (f) against A549 cells in normoxia incubated for 24 h with/without irradiation.\nCytotoxicity and phototoxicity of 6d against HT29 (g) and 4T1 (h) cells in normoxia, and against A549 (i), HT29 (j), and 4T1 (k) cells in hypoxia incubated for 24 h\nwith/without irradiation. L: Light. Light dose: 520 nm, 200 mW/cm2, 10 min. The related data are presented as the means \u00b1 SD of three separate assays, n = 3. (*, P\n< 0.05; **, P < 0.01; ***, P < 0.001).\n\n\n2.4. Subcellular localization studies correlation coefficient (PCC) of 0.83 and 0.85, respectively. Similarly,\n we investigated the subcellular distribution of 6a in 4T1 cells. Micro-\n The subcellular localization of 6d in A549 cells was determined by scopic images demonstrated a notable correlation with PCC values of\nconfocal laser scanning microscopy (CLSM). As depicted in Fig. 6, the 0.85 and 0.81 with Mito-Tracker Green and Lyso-Tracker Green,\nred fluorescence emitted by 6d exhibited a significant overlap with the respectively. These experimental results indicate mitochondria and\ngreen fluorescence channels of the commercial mitochondrial dye Mito- lysosomal as the primary localization of the Ru(II) complexes we\nTracker Green and lysosomal dye Lyso-Tracker Green, with a Pearson\u2019s designed.\n\n\n 5\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n\n\nFig. 5. (a) CLSM images of 4T1 cells upon incubation with 6a (10 \u03bcM) and calcein-AM (live, green)/PI (dead, red) in the dark or upon irradiation. (b) Ratio of green/\nred fluorescence intensity of (a). (c) CLSM images of A549 cells upon incubation with 6d (10 \u03bcM) and calcein-AM (live, green)/PI (dead, red) in the dark or upon\nirradiation. (d) Ratio of green/red fluorescence intensity of (c). D: Dark; L: Light. Light dose: 520 nm, 200 mW/cm2, 10 min. Scale bar: 100 \u03bcm. The related data are\npresented as the means \u00b1 SD of three separate assays. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).\n\n\n\n\n2.5. Intracellular ROS generation depolarization is indicated by a decreased red/green fluorescence in-\n tensity ratio (I585nm/I530nm). Both A549 and 4T1 cells showed a decrease\n Considering the remarkable phototoxicity performed by 6a and 6d, in red fluorescence signal and a significant increase in green fluores-\nwe postulated that this effect might be attributed to their ability to cence intensity upon illumination after incubation with 6a/6d, respec-\ninduce a substantial amount of ROS. The intracellular ROS levels tively (Fig. 8). Notably, the ratio of green to red fluorescence of 4T1 cells\ninduced by complexes 6a and 6d were examined through 2,7-dichloro- and A549 cells increased by 2.44-fold and 3.26-fold, respectively. These\nfluorescin diacetate (DCFH-DA) fluorescence assay measured by findings provide evidence that 6a and 6d likely induce cell death by\nconfocal microscopy. DCFH-DA is a non-fluorescent dye that can be damaging the mitochondria.\nenzymatically hydrolyzed and oxidized by ROS to form fluorescent Based on the aforementioned experimental findings, it can be\ndichlorofluorescein (DCF), emitting a green fluorescence. The fluores- inferred that complexes 6a and 6d possess the ability to accumulate\ncence images disclosed that a significantly higher intensity (3.8 times within mitochondria upon entering tumor cells. Upon light exposure,\ngreater than that of the control group) in A549 cells incubated with 6d these complexes generate ROS, leading to mitochondrial dysfunction\nupon irradiation. In contrast, negligible green fluorescence was and triggering tumor cell death, thereby exhibiting the photodynamic\nobserved in the other groups, which either received light treatment therapeutic efficacy.\nalone or were incubated with only 6d (Fig. 7c and d). Similar trends\nwere observed in 4T1 cells incubated with 6a, with a 2.1-fold increase in\nfluorescence intensity compared to the control group (Fig. 7a and b). 2.7. The mechanisms of cell death\nThese findings unequivocally verify that 6a and 6d possess the capa-\nbility to generate substantial levels of ROS within cells under specific Ferroptosis, a recently unveiled mode of regulated cell death, is\nlight conditions, thereby validating their excellent phototoxicity. attributed to the buildup of lipid peroxides (LPOs) mediated by intra-\n cellular iron, and has garnered increasing attention due to its link with\n immune responses in tumors [39,40]. The induction of ferroptosis,\n2.6. Mitochondria damage characterized by the depletion of glutathione (GSH), downregulation of\n glutathione peroxidase 4 (GPX4), and accumulation of LPOs, is widely\n The generation of ROS typically results in an imbalance in the recognized as a promising therapeutic strategy in cancer treatment\ncellular redox environment, commonly indicated by damage to the [41\u201343].\nintegrity of the mitochondrial membrane [37,38]. Maintaining mito- Leveraging knowledge of the mechanisms, the impact of 6d on these\nchondrial membrane integrity is crucial for cell survival. Since com- distinctive ferroptosis-associated hallmarks was further explored in\nplexes 6a and 6d primarily localize in the mitochondria, their impact on A549 cells. Measurement of the GSH level was carried out using\nmitochondrial integrity was assessed by monitoring changes in mito- Glutathione Assay Kit to evaluated the GSH depletion capability of 6d.\nchondrial membrane potential (MMP) using the commercial probe JC-1, Remarkably, a substantial decrease in GSH levels was observed in A549\nwhich selectively stains polarized mitochondria. Mitochondrial cells incubated with 6d upon irradiation, likely attributed to the\n\n 6\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n\n\nFig. 6. Subcellular localization of 6a and 6d. CLSM images (a) and Z-stack scanning (b) of 4T1 cells after incubated with Mito-Tracker Green (2 \u03bcM) and 6a (10 \u03bcM).\n(c) The Pearson Correlation Coefficient of 6a and Mito-Tracker Green in 4T1 cells. CLSM images (d) and Z-stack scanning (e) of 4T1 cells after incubated with Lyso-\ntracker Green (2 \u03bcM) and 6a (10 \u03bcM). (f) The Pearson Correlation Coefficient of 6a and Lyso-tracker Green in 4T1 cells. CLSM images (g) and Z-stack scanning (h) of\nA549 cells after incubated with Mito-Tracker Green (2 \u03bcM) and 6d (10 \u03bcM). (i) The Pearson Correlation Coefficient of 6d and Mito-Tracker Green in A549 cells. CLSM\nimages (j) and Z-stack scanning (k) of A549 cells after incubated with Lyso-tracker Green (2 \u03bcM) and 6d (10 \u03bcM). (l) The Pearson Correlation Coefficient of 6d and\nLyso-tracker Green in A549 cells. Scale bar: 25 \u03bcm.\n\n\n\n\noxidation process catalyzed by ROS. In contrast, the reduction in GSH Z-VAD-FMK (a pancaspase inhibitor). The pre-incubation with 3-MA and\nlevels in the remaining experimental groups was almost negligible Fer-1 demonstrated significant mitigating effects on cell death induced\n(Fig. 9a). Previous research has proposed that reduced glutathione levels by treatment with 6d and exposure to irradiation. Notably, Fer-1\nmay impede the expression of GPX4, given that glutathione serves as the exhibited a more pronounced impact than 3-MA. These findings\nintrinsic substrate for GPX4. Subsequent Western blot analysis validated strongly indicate that 6d primarily triggers cell demise through the in-\na decrease in GPX4 expression following treatment with 6d and expo- duction of ferroptosis, rather than apoptosis or necrosis.\nsure to light (Fig. 9c). Furthermore, the Lipid Peroxidation MDA Assay In consideration of previous research suggesting a potential link\nKit was employed to examine the intracellular generation of malon- between ferroptosis and immunogenic cell death (ICD), we sought to\ndialdehyde (MDA), a crucial end product of lipid peroxidation known explore the impact of 6d on this particular mode of cell demise. ICD\nfor its positive regulatory impact on ferroptosis [44]. While a certain involves the translocation of endoplasmic reticulum-resident calreticu-\nlevel of MDA was produced upon treatment with 6d alone, a significant lin (CRT) and heat shock protein 70 (HSP70) to the cell surface, along\nenhancement of the MDA generation was observed upon exposure to with the release of adenosine triphosphate (ATP) and the nuclear high-\nlight in A549. Specifically, intracellular MDA levels increased by mobility group box 1 (HMGB1) protein [45\u201347]. These characteristic\n7.77-fold following treatment with 6d in conjunction with irradiation, markers were therefore investigated upon treatment of A549 cells with\ncompared to the PBS + light group (Fig. 9b). Notably, the addition of 6d.\nFer-1 (ferrostatin-1, a ferroptosis inhibitor) effectively alleviated MDA Utilizing immunofluorescence confocal laser scanning microscopy,\ngeneration. These combined findings provide compelling evidence that we discerned the migration of CRT, as depicted in Fig. 9e. Notably, no\nthe combination of 6d treatment and exposure to light irradiation in- discernible alterations were noted following treatment with PBS or DOX.\nduces cell death through the process of ferroptosis. Next, the In stark contrast, a notable translocation of CRT to the cytoplasm or cell\nC11-BODIPY 581/591 probe was employed to measure the level of lipid membrane was evident upon treatment with 6d and exposure to light,\nROS in A549 cells. It was observed that the cells treated with 6d with the CRT ratio reaching 3.9-fold that of the PBS + light group\nexhibited a subtle shift in fluorescence from red to green. However, this (Fig. 9g). Additionally, the migration of nuclear HMGB1 protein from\ntrend was significantly enhanced in the light-exposed group, indicating the nucleus into the extracellular space following treatment with 6d\nthat 6d under light conditions markedly induces a substantial increase in upon irradiation was confirmed, with the nuclear HMGB1 level\nendogenous lipid ROS levels within the cells (Fig. S9). decreased to 12.9 % of the PBS + light group (Fig. 9f and h). Subse-\n Several specific inhibitors were employed to further understand the quently, we investigated the extracellular secretion of ATP using a\ncontribution of ferroptosis in comparison to traditional cell death specific bioluminescence detection kit. Remarkably, a remarkable 5-fold\nmechanism (Fig. 9d), including 3-methyladenine (3-MA, an autophagy increase in extracellular ATP levels was detected in A549 cells treated\ninhibitor), Fer-1, necrostatin-1 (Nec-1, an inhibitor of necroptosis) and with 6d and subsequently exposed to irradiation, as illustrated in Fig. 9i.\n\n\n 7\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n\n\nFig. 7. Intracellular ROS generation by 6a and 6d. CLSM images of intracellular ROS levels in 4T1 cells treated with 6a (10 \u03bcM) (a) and A549 cells treated with 6d\n(10 \u03bcM) (c) with/without a 520 nm laser irradiation (200 mW/cm2, 10 min). Average fluorescence intensity of DCFH-DA in 4T1 cells (b) and A549 cells (d) of\ndifferent groups. D: Dark; L: Light. Light dose: 520 nm, 200 mW/cm2, 10 min. Scale bar: 100 \u03bcm. The related data are presented as the means \u00b1 SD of three separate\nassays. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).\n\n\n\n\nFig. 8. Induction of Mitochondria Damage by 6a and 6d. CLSM images of MMP condition in 4T1 cells treated with 6a (10 \u03bcM) (a) and A549 cells treated with 6d (10\n\u03bcM) (c) with/without laser irradiation (520 nm, 200 mW/cm2, 10 min) measured with JC-1 dye. Ratio of green/red fluorescence intensity of 4T1 cells (b) and A549\ncells (d). D: Dark; L: Light. Light dose: 520 nm, 200 mW/cm2, 10 min. Scale bar: 100 \u03bcm. The related data are presented as the means \u00b1 SD of three separate assays.\n(*, P < 0.05; **, P < 0.01; ***, P < 0.001).\n\n\n\n 8\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n\n\nFig. 9. The mechanisms of cell death induced by 6d. (a) Intracellular GSH content of A549 cells after treatment with PBS, RuB (10 \u03bcM) or 6d (10 \u03bcM) with/without\nirradiation. (b) Intracellular MDA content of A549 cells after treatment with PBS, RuB (10 \u03bcM), 6d (10 \u03bcM) or 6d (10 \u03bcM) + Fer-1 (1 \u03bcM) with/without irradiation.\n(c) Intracellular GPX4 protein levels by Western blotting assay and MDA content for cells treated with 6d. (d) Cell viabilities of A549 cells after coincubation for 24 h\nwith 6d (10 \u03bcM) in the presence of different inhibitors, including 3-MA (100 \u03bcM), Fer-1 (1 \u03bcM), Nec-1 (50 \u03bcM) and Z-VAD-FMK (5 \u03bcM) with/without irradiation.\nEvaluation for hallmarks of ICD in A549 cells upon treatment with DOX (10 \u03bcM) and 6d (10 \u03bcM) with/without irradiation. Immunofluorescence confocal laser\nscanning microscopy stained with (e) calreticulin-specific antibody and (f) HMGB1-specific antibody. (g) Quantized data of (e). (h) Quantized data of (f). (i) Release\nof ATP into the cell culture supernatant. D: Dark; L: Light. Light dose: 520 nm, 200 mW/cm2, 10 min. Scale bar: 100 \u03bcm. The related data are presented as the means\n\u00b1 SD of three separate assays. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).\n\n\n\n\nCollectively, these findings strongly suggest the potential of 6d to induce groups, highlighting the potent in vivo antitumor efficacy of 6d\nICD upon light irradiation. (Fig. 10c).\n Hematoxylin and eosin (H&E) staining results revealed severe\n damage to tumor cells in the 6d + light group, whereas only slightly\n2.8. Antitumor activity in vivo damage was observed in the RuB + light group (Fig. 10f). No damage or\n necrosis was detected in the tumor tissues of the remaining two groups.\n Given the remarkable in vitro performance of 6d, its antitumor effi- These results validate the remarkable in vivo photodynamic therapy and\ncacy was further evaluated in A549 tumor-bearing BALB/c nude mice anti-tumor activity of 6d. Furthermore, the mice exhibited normal\nmodel. Upon the establishment of solid tumors, the mice models were behavior without any signs of pain, stress, or discomfort, and the body\nrandomly assigned to four groups: Group 1 (PBS only), Group 2 (RuB + weight of the 6d + Light group remained unchanged (Fig. 10e).\nlight), Group 3 (6d only), and Group 4 (6d + light), each comprising five Consistently, no pathological abnormalities were observed in the H&E\nnude mice. Six hours following intravenous injection, the mice were staining of vital organs and blood routine indexes, indicating the high\neither kept in the dark or exposed to irradiation (520 nm, 200 mW/cm2, biocompatibility of the treatment (Fig. 10f and S10). Utilizing 4T1\n10 min). The tumor volume and body weight of each mouse was murine models and identical methodologies, a parallel assessment was\nmeasured and recorded every three days over a span of 12 days undertaken to evaluate the in vivo anti-tumor efficacy and biological\n(Fig. 10a). Under laser irradiation, complex 6d nearly completely safety of 6a. The outcomes revealed a remarkable suppression of 85.8 %\ninhibited tumor growth, resulting in a reduction of tumor volume and in tumor volume and an impressive 92.73 % reduction in tumor weight\nweight by 85.9 % and 95.36 % respectively, outperforming the rates of (Fig. 10g\u2212 l). In conclusion, the research findings demonstrated signifi-\nRuB + light group (0.79 % and 77.54 %) (Fig. 10b\u2212 d). These findings cant potential for 6d and 6a to reduce systemic toxicity and exert anti-\nwere further supported by isolated tumor images from the different\n\n\n\n\n 9\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n\n\nFig. 10. Evaluation of the treatment in 4T1/A549 tumor-bearing BALB/c nude mice model after intratumor injection of 6a/d (5 mg/kg) and exposure to irradiation.\n(a) and (g) Schematic illustration of the treatment. (b) and (h) Tumor growth curves of the tumor-bearing mice during different treatments. (c) and (i) Images of the\ncollected tumors from tumor-bearing mice 12 days after different treatment. (d) and (j) Means tumor weight of mice after different treatments. (e) and (k) Body\nweights of the mice during different treatments. (f) and (l) Images of H&E staining heart, liver, spleen, lung, kidney and tumor slices from mice after different\ntreatments. L: Light. Light dose: 520 nm, 200 mW/cm2, 10 min. Scale bar: 100 \u03bcm. The related data are presented as the means \u00b1 SD of three separate assays. (*, P <\n0.05; **, P < 0.01; ***, P < 0.001).\n\n\ntumor effects through various mechanisms, emphasizing the need for potential of 6d as a novel PDT agent for challenging lung cancer.\nfurther thorough exploration in future studies.\n 4. Experimental\n3. Conclusion\n 4.1. Materials and reagents\n In brief, this article discusses the synthesis of five structurally\nstraightforward Ru(II) metal complexes 6a-e featuring bipyridyl ligands All chemical reagents and solvents for synthesis were obtained from\nfor PDT, as well as their photo-physicochemical properties. These qualified reagent supplies (Aladdin, Bidepharm and Meryer) with\ncomplexes were found to exhibit a maximum absorption peak at analytical reagent grade and were used in whole experiment without\napproximately 460 nm, with emission wavelengths extending to 650 further purification. All reactions were monitoring in real time by thin-\nnm. Notably, the most promising Ru(II) complex 6d displayed layer chromatography (TLC). Reaction products were isolated and pu-\noutstanding photodynamic properties, characterized by a high 1O2 rified by silica gel column chromatography. 1H NMR and 13C NMR\nquantum yield and remarkable \u2022O2\u2212 generation capacity. It was spectra were recorded on a Bruker AV 400 M spectrometer with an in-\nobserved that 6d demonstrates limited cytotoxicity against various ternal standard (TMS). UV\u2013vis and fluorescence spectra were performed\ntumor cell lines in the dark. However, when exposed to 520 nm light, its on a Lambda 35 UV\u2013visible spectrophotometer and RF-5301PC fluo-\ncytotoxic effects are significantly enhanced, achieving an IC50 value of rescence spectrophotometer, respectively. Confocal imaging of cells was\n5.66\u20137.17 \u03bcM against three cancer cell lines, with PI exceeding 14. performed using a confocal microscope (ZISS M880, Germany) imaging\nAdditionally, 6d exhibits strong phototoxicity even in hypoxic tumor system. Complexes 6a-e were checked with high-performance liquid\nenvironments (IC50s = 4.16\u2212 9.12 \u03bcM), indicating its potential as Type- chromatography (HPLC) with a purity of >95 % (Fig. S41\u2212 45). Com-\nI/-II photosensitizer. Further mechanistic studies have shown that 6d pounds 2\u20134 were synthesized according literatures [48].\nsignificantly increases ROS generation in cells, leading to mitochondrial DCFH, DHE, HPF, DPBF were purchased from Bide Pharmatech\ndamage and subsequent ferroptosis. Interestingly, it has been demon- (China). MTT was purchased from Energy Chemical (China). Calcein\nstrated that 6d also induces ICD, triggering the release of damage- AM/PI Double Stain Kit was purchased from were purchased from\nassociated molecular patterns (DAMPs). In vivo experiments further Yeasen Biotechnology (China). Lyso-Tracker Green and Mito-tracker\nconfirmed the high photodynamic activity and anti-tumor efficacy of 6d. Green was purchased from Beyotime Biotechnology (China). 2\u2032,7\u2032-\nCollectively, the multifaceted mechanisms underscore the promising dichlorofluorescein diacetate (DCFH-DA) kit were purchased from\n\n\n 10\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\nThermo Scientific (USA). Mitochondrial Membrane Potential Assay Kit 127.15, 124.71, 123.95, 122.07, 121.51, 62.93, 61.89, 53.06, 52.92,\nwith JC-1 was purchased from Abbkine Biotechnology (China). GSH 21.21. HRMS(ESI) m/z Calcd for C23H26N4 [M + H]+, 359.2236; found,\nAssay Kit was purchased from Abcam (UK). MDA Assay Kit was pur- 359.2237.\nchased from Abnova (China). Calreticulin (D3E6) XP\u00ae Rabbit mAb,\nHMGB1 Antibody, Anti-rabbit IgG (H + L), F (ab\u2019)2 Fragment (Alexa 4.2.5. Preparation of compound 5e\nFluor\u00ae 647 Conjugate), and DAPI were purchased from Cell Signaling Referring to the method of compound 5a, 4-methylpiperidine was\nTechnology (USA). replaced with 1-phenylpiperazine to obtain 4-methyl-4\u2019-((4-phenyl-\n piperazin-1-yl)methyl)-2,2\u2032-bipyridine (5e) as yellow solid (71 %). 1H\n4.2. Synthesis of ligands and complexes NMR (400 MHz, CDCl3) \u03b4 8.64 (d, J = 5.0 Hz, 1H, ArH), 8.55 (d, J = 5.0\n Hz, 1H, ArH), 8.35 (s, 1H, ArH), 8.24 (s, 1H, ArH), 7.41 (s, 1H, ArH),\n4.2.1. Preparation of compound 5a 7.31\u20137.22 (m, 2H, 2ArH), 7.15 (d, J = 3.3 Hz, 1H, ArH), 6.93 (d, J = 8.2\n To a mixed solution of compound 4 (1.048 g, 4 mmol), 4-methylpi- Hz, 2H, 2ArH), 6.86 (m, 1H, ArH), 3.67 (s, 2H, CH2), 3.23 (m, 4H,\nperidine (0.372 g, 4 mmol) and K2CO3 (0.553 g, 4 mmol) in MeCN 2CH2), 2.67 (m, 4H, 2CH2), 2.45 (s, 3H, CH3). 13C NMR (101 MHz,\n(20 mL) was added 1\u20132 drops of DMF. After stirring at 85 \u25e6 C for 12 h, the CDCl3) \u03b4 156.40, 155.93, 151.28, 149.28, 149.03, 148.22, 129.13,\nsolution was extracted with DCM (40 mL \u00d7 3). The combined organic 124.79, 123.98, 122.09, 121.54, 119.78, 116.13, 61.94, 53.24, 49.12,\nlayers were dried with anhydrous Na2SO4 and evaporated to afford 21.24. HRMS(ESI) m/z Calcd for C22H24N4 [M + H]+, 345.2079; found,\ncrude product, which was finally purified by column chroma-tography 345.2078.\nusing ammonia MeOH/DCM (1:15, v/v) as the eluent to afford 4-\nmethyl-4\u2019-((4-methylpiperidin-1-yl)methyl)-2,2\u2032-bipyridine (5a) as light 4.2.6. Preparation of complex 6a\nyellow solid (0.933 g, 83 %). 1H NMR (400 MHz, CDCl3) \u03b4 8.61 (d, J = A solution of 5a (0.421 g, 1.5 mmol) and RuCl3\u22c5H2O (0.103 g, 0.5\n5.0 Hz, 1H, ArH), 8.54 (d, J = 4.9 Hz, 1H, ArH), 8.29 (d, J = 1.6 Hz, 1H, mmol) in ethylene glycol (5 mL) was stirred for 6 h at 120 \u25e6 C. Then three\nArH), 8.22 (d, J = 1.7 Hz, 1H, ArH), 7.36 (dd, J = 5.0, 1.7 Hz, 1H, ArH), steaming water (40 mL) and NH4PF6 (0.744 g, 4 mmol) were added and\n7.13 (dd, J = 5.0, 1.7 Hz, 1H, ArH), 3.56 (s, 2H, CH2), 2.85 (m, 2H, CH2), then stirred at room temperature for another 10 min. The reaction was\n2.44 (s, 3H, CH3), 2.01 (m, 2H, CH2), 1.60 (m, 2H, CH2), 1.28 (m, 3H, filtered and washed with three steaming water (5 mL \u00d7 3) and chloro-\nCH2, CH), 0.92 (d, J = 6.1 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) \u03b4 form (10 mL \u00d7 3). The crude product was purified by column chro-\n156.16, 156.08, 149.31, 149.10, 148.98, 148.12, 124.64, 123.98, matography with ammonia MeOH/DCM (1:20, v/v) as the eluent to\n122.06, 121.54, 62.42, 54.12, 34.29, 30.64, 21.90, 21.20. HRMS(ESI) obtain target compound 6a as orange-red solid (0.321 g, 52 %). 1H NMR\nm/z Calcd for C18H23N3 [M + H]+, 282.1970; found, 282.1962. (400 MHz, DMSO\u2011d6) \u03b4 8.68 (s, 6H, 6ArH), 7.74\u20137.61 (m, 3H, 3ArH),\n 7.59\u20137.51 (m, 3H, 3ArH), 7.46 (d, J = 5.8 Hz, 3H, 3ArH), 7.39\u20137.33 (m,\n4.2.2. Preparation of compound 5b 3H, 3ArH), 3.66 (s, 6H, 3CH2), 2.97\u20132.61 (m, 6H, 3CH2), 2.53 (s, 9H,\n Referring to the method of compound 5a, 4-methylpiperidine was 3CH3), 1.59 (s, 6H, 3CH2), 1.43\u20131.29 (m, 6H, 3CH2), 1.22 (s, 9H, 3CH2,\nreplaced with 1,4\u2032-bipiperidine to obtain 1\u2019-((4\u2032-methyl-[2,2\u2032-bipyridin]- 3CH), 0.90 (d, J = 6.2 Hz, 9H, 3CH3). 13C NMR (101 MHz, DMSO\u2011d6) \u03b4\n4-yl)methyl)-1,4\u2032-bipiperidine (5b) as light yellow solid (80 %). 1H NMR 156.90, 156.53, 151.33, 150.75, 149.97, 129.01, 125.57, 124.29, 53.89,\n(400 MHz, CDCl3) \u03b4 8.53 (d, J = 5.0 Hz, 1H, ArH), 8.46 (d, J = 5.0 Hz, 34.24, 30.40, 22.22, 21.15. HRMS(ESI) m/z Calcd for C54H69N9Ru [M-\n1H, ArH), 8.22 (d, J = 1.6 Hz, 1H, ArH), 8.15 (d, J = 1.6 Hz, 1H, ArH), 2PF6]2+, 472.7360; found, 472.7365.\n7.26 (m, 1H, ArH), 7.05 (m, 1H, ArH), 3.48 (s, 2H, CH2), 2.86 (m, 2H,\nCH2), 2.42 (m, 4H, 2CH2), 2.36 (s, 3H, CH3), 2.17 (m, 1H, CH), 1.94 (m, 4.2.7. Preparation of complex 6b\n2H, CH2), 1.69 (m, 2H, CH2), 1.60\u20131.47 (m, 6H, 3CH2), 1.35 (m, 2H, Referring to the method of compound 6a, 5a was replaced with 5b to\nCH2). 13C NMR (101 MHz, CDCl3) \u03b4 156.22, 156.04, 149.25, 149.12, obtain target compound 6b as orange-red solid (55 %). 1H NMR (400\n148.98, 148.10, 124.65, 123.82, 122.04, 121.39, 62.62, 61.97, 53.65, MHz, DMSO\u2011d6) \u03b4 8.77\u20138.57 (m, 6H, 6ArH), 7.70\u20137.48 (m, 6H, 6ArH),\n50.23, 27.91, 26.42, 24.81, 21.19. HRMS(ESI) m/z Calcd for C22H30N4 7.48\u20137.41 (m, 3H, 3ArH), 7.39\u20137.32 (m, 3H, 3ArH), 3.69 (s, 6H, 3CH2),\n[M + H]+, 351.2549; found, 351.2544. 3.19 (d, J = 12.6 Hz, 3H, 3CH), 3.03\u20132.83 (m, 12H, 6CH2), 2.52 (s, 9H,\n 3CH3), 2.16\u20131.61 (m, 36H, 18CH2), 1.52\u20131.28 (m, 6H, 3CH2). 13C NMR\n4.2.3. Preparation of compound 5c (101 MHz, DMSO\u2011d6) \u03b4 156.92, 156.88, 156.45, 151.26, 150.71, 150.01,\n Referring to the method of compound 5a, 4-methylpiperidine was 129.03, 127.58, 125.50, 124.19, 62.98, 59.98, 52.01, 49.60, 26.16,\nreplaced with piperidin-4-one to obtain 1-((4\u2032-methyl-[2,2\u2032-bipyridin]-4- 23.33, 21.87, 21.11. HRMS(ESI) m/z Calcd for C66H90N12Ru [M-\nyl)methyl)piperidin-4-one (5c) as light yellow solid (77 %). 1H NMR 2PF6]2+, 576.3228; found, 576.3239.\n(400 MHz, CDCl3) \u03b4 8.57 (d, J = 5.0 Hz, 1H, ArH), 8.47 (d, J = 5.0 Hz,\n1H, ArH), 8.30 (d, J = 1.6 Hz, 1H, ArH), 8.17 (d, J = 1.7 Hz, 1H, ArH), 4.2.8. Preparation of complex 6c\n7.31 (dd, J = 4.9, 1.7 Hz, 1H, ArH), 7.08 (dd, J = 5.0, 1.7 Hz, 1H, ArH), Referring to the method of compound 6a, 5a was replaced with 5c to\n3.64 (s, 2H, CH2), 2.72 (m, 4H, 2CH2), 2.42 (m, 4H, 2CH2), 2.37 (s, 3H, obtain target compound 6c as orange-red solid (53 %). 1H NMR (400\nCH3). 13C NMR (101 MHz, CDCl3) \u03b4 207.72, 155.50, 154.79, 148.30, MHz, DMSO\u2011d6) \u03b4 8.73\u20138.69 (m, 3H, 3ArH), 8.68\u20138.65 (m, 3H, 3ArH),\n147.98, 147.55, 147.22, 123.80, 122.55, 121.06, 120.13, 59.94, 52.12, 7.70\u20137.51 (m, 6H, 6ArH), 7.48 (d, J = 5.9 Hz, 3H, 3ArH), 7.38\u20137.34 (m,\n40.23, 20.18. HRMS(ESI) m/z Calcd for C17H19N3O [M + H]+, 3H, 3ArH), 3.86 (s, 12H, 6CH2), 3.72 (s, 6H, 3CH2), 2.53 (s, 9H, 3CH3),\n282.1606; found, 282.1601. 1.67 (d, J = 5.6 Hz, 12H, 6CH2). 13C NMR (101 MHz, DMSO\u2011d6) \u03b4\n 156.89, 156.51, 151.27, 150.70, 149.96, 129.00, 127.52, 125.56,\n4.2.4. Preparation of compound 5d 124.07, 106.59, 64.09, 60.16, 51.50, 34.81, 21.12. HRMS(ESI) m/z\n Referring to the method of compound 5a, 4-methylpiperidine was Calcd for C51H57N9O3Ru [M-2PF6 + 3CH3CHO]2+, 538.7207; found,\nreplaced with 1-benzylpiperazine to obtain 4-((4-benzylpiperazin-1-yl) 538.7210.\nmethyl)-4\u2032-methyl-2,2\u2032-bipyridine (5d) as yellow solid (74 %). 1H NMR\n(400 MHz, CDCl3) \u03b4 8.60 (d, J = 5.0 Hz, 1H, ArH), 8.54 (d, J = 5.0 Hz, 4.2.9. Preparation of complex 6d\n1H, ArH), 8.30 (d, J = 1.6 Hz, 1H, ArH), 8.22 (d, J = 1.7 Hz, 1H, ArH), Referring to the method of compound 6a, 5a was replaced with 5d to\n7.36\u20137.31 (m, 4H, 4ArH), 7.25 (m, 2H, 2ArH), 7.13 (dd, J = 5.0, 1.7 Hz, obtain target compound 6d as orange-red solid (51 %). 1H NMR (400\n1H, ArH), 3.59 (s, 2H, CH2), 3.54 (s, 2H, CH2), 2.48 (m, 11H, CH3, MHz, DMSO\u2011d6) \u03b4 8.79\u20138.61 (m, 6H, 6ArH), 7.70\u20137.48 (m, 6H, 6ArH),\n4CH2). 13C NMR (101 MHz, CDCl3) \u03b4 156.29, 155.98, 149.16, 148.99, 7.44 (d, J = 6.0 Hz, 3H, 3ArH), 7.38\u20137.21 (m, 18H, 18ArH), 3.67 (d, J =\n148.69, 148.15, 137.70, 129.33, 129.08, 128.38, 128.25, 127.36, 3.5 Hz, 6H, 3CH2), 3.55\u20133.41 (m, 6H, 3CH2), 2.51 (s, 12H, 6CH2), 2.42\n\n 11\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n(s, 21H, 3CH3, 3CH2). 13C NMR (101 MHz, DMSO\u2011d6) \u03b4 156.86, 156.50, 4.7. Calculation of fluorescence quantum yield (\u03a6F)\n151.26, 150.71, 150.39, 149.94, 129.33, 129.01, 128.66, 127.67,\n127.46, 125.56, 124.29, 62.48, 60.66, 53.25, 52.87, 21.14. HRMS(ESI) Using RuB (\u03a6F = 0.328, in a mixture of methanol and ethanol (1:4,\nm/z Calcd for C69H78N12Ru [M-2PF6]2+, 588.2758; found, 588.2777. v/v)) as a reference, \u03a6F of 6a and 6d were determined in the same\n solvent. \u03a6F was calculated according to the following equation 3 re-\n4.2.10. Preparation of complex 6e ported in the literature [36].\n Referring to the method of compound 6a, 5a was replaced with 5e to\n \u03a6F(X) = \u03a6F(S) (ASFX/AXFS) (nX/nS)2\nobtain target compound 6e as orange-red solid (57 %). 1H NMR (400\nMHz, DMSO\u2011d6) \u03b4 8.79\u20138.67 (m, 6H, 6ArH), 7.75\u20137.49 (m, 9H, 9ArH), where F denotes the integrated area of fluorescence spectrum at the\n7.37 (d, J = 5.8 Hz, 3H, 3ArH), 7.25\u20137.15 (m, 6H, 6ArH), 6.98\u20136.90 (m, same excitation wavelength. A denotes the absorbance values at the\n6H, 6ArH), 6.82\u20136.73 (m, 3H, 3ArH), 3.80\u20133.67 (m, 6H, 3CH2), excitation wavelength. N denotes the refractive index of the solvents. S\n3.23\u20133.11 (m, 12H, 6CH2), 2.66\u20132.56 (m, 12H, 6CH2), 2.53 (s, 9H, denotes the samples awaiting testing, and X denotes the reference, that\n3CH3). 13C NMR (101 MHz, DMSO\u2011d6) \u03b4 156.94, 156.55, 151.39, is, RuB. (nX/nS = 1 in this work).\n150.78, 150.28, 149.99, 129.42, 129.05, 127.77, 125.63, 124.33,\n119.41, 115.88, 60.64, 53.21, 48.60, 21.15. HRMS(ESI) m/z Calcd for\n 4.8. Photostability study\nC66H72N12Ru [M-2PF6]2+, 567.2523; found, 567.2528.\n The stock solution of 6a/6d in DMSO (5 mM) was appropriately\n4.3. Lipophilicity diluted with methanol to achieve the desired working concentration (20\n \u03bcM). The UV\u2013vis absorption spectrum was measured at different irra-\n The lipophilicity of RuB and 6a-e was assessed through the deter- diation (520 nm, 200 mW/cm2) times.\nmination of their octanol-water partition coefficients, denoted as log P,\nemploying a previously documented methodology [49]. 4.9. Electron paramagnetic resonance (EPR) assay\n\n4.4. The absorption and fluorescence spectra The EPR assay were conducted using an electron spin resonance\n spectrometer at 298 K. The type of ROS was identified using the \u2022O\u22122\n The stock solution of 6a-e in DMSO (5 mM) was appropriately scavenger 5,5-dimethyl-1-pyrroline N-oxide (DMPO), \u2022OH scavenger\ndiluted with methanol to achieve the desired working concentration (10 2,2,6,6-tetramethyl-4-nitropiperidine oxide (TNPO) and 1O2 scavenger\n\u03bcM). Spectral scanning was carried out using 3 mL of the prepared so- 2,2,6,6\u2013tetramethylpiperidine (TEMP). Light source: 10 min, 520 nm,\nlution in cuvettes. For the emission spectrum scans, the complexes were and 200 mW/cm2.\nexcited at a wavelength of 460 nm.\n 4.10. Cell culture and in vitro cytotoxicity assay\n 2\u2212\n4.5. In vitro ROS, \u2022O , and \u2022OH detection\n All cell lines used in this study, including HT29, A549, and 4T1, were\n Firstly, prepare stock solutions (5 mM) of DCFH or 6a-e in DMSO. sourced from the Shanghai Institute of Cell Biology (China). All cells\nThen, add a specific volume of each stock solution into 3 mL of methanol were cultured under carefully controlled conditions at a constant tem-\nto achieve a uniform concentration of 10 \u03bcM for each. Then, the probe perature of 37 \u25e6 C with a 5 % CO2 atmosphere. They were routinely\nDCFH-DA, DHE, or HPF was added to the methanol solution to achieve incubated in dulbecco\u2019s modified eagle medium (DMEM) that was\nthe desired working concentration (10 \u03bcM). All procedures are con- supplemented with 10 % fetal bovine serum (FBS).\nducted under light-protected conditions. The fluorescence spectrum was The MTT assay was employed to determine the in vitro cytotoxicities\nmeasured at different irradiation (520 nm, 200 mW/cm2) time. of complexes 6a-e. HT29, A549, and 4T1 cells were seeded in 96-well\n microplate at a density of 1 \u00d7 105 cells/mL in 100 \u03bcL culture medium\n containing 10 % FBS and incubated for 12 h. Different concentrations (2,\n4.6. Calculation of 1O2 quantum yield (\u03a6\u0394) 5, 10, 20, 40, and 100 \u03bcM) of the complexes were added individually to\n the culture media and incubated for another 12 h under normoxic or\n DPBF was utilized as the detection agent for the measurement of the hypoxic condition. The supernatant was replaced with a fresh culture\n\u03a6\u0394. The absorbance of DPBF in methanol was adjusted to approximately medium, and the cells were subjected to irradiation (520 nm, 200 mW/\n1.0 at 415 nm. To minimize the 1O2 quenching by RuB and 6a-e cm2) for 10 min, and incubated for an additional 24 h in the dark. Cells\nthemselves, their absorbance at 520 nm were adjusted to below 0.1. All in the non-irradiation group were replaced with a fresh culture medium\nprocedures are conducted under light-protected conditions. The and maintained in the dark. MTT solution (10 \u03bcL, 5 mg/mL in PBS) was\ndecomposition rates of DPBF by the Ru(II) complexes were monitored at added to each well, and the cells were further incubated for 3 h. After-\ndifferent irradiation (520 nm, 200 mW/cm2) times, with the absorbance ward, the culture medium were removed and 100 \u03bcL of DMSO solution\nchanges of DPBF at 412 nm utilized for quantification of \u03a6\u0394. Using RuB was added to each well to dissolve the formazan crystals. The absor-\n(\u03a6\u0394 = 0.73, in methanol) as a reference. The \u03a6\u0394 of the samples were bance values at 570 nm were then measured using a multifunction\ncalculated according to the following equation: microplate reader, and the corresponding cell viability was determined\n\u03a6\u0394(X) = \u03a6\u0394(S) (kX \u00d7 FS) / (kS \u00d7 FX) based on the data obtained.\n\nwhere k denotes the calibrated slope of the linear fit of the cumulative 4.11. Live/dead cell stain\ndecrease of the absorbance at 415 nm versus the change of irradiation\ntime. F denotes the correction factor calculated using equation 2. S de- Cells were seeded on confocal dishes at a density of 1 \u00d7 105 cells/mL\nnotes the samples awaiting testing, and X denotes the reference, that is, in 2.0 mL culture medium and allowed to adhere upon incubation for 12\nRuB. h. Following removal of the culture medium, the cells were subjected to\n various treatments: (control, L, 6d/6a (10 \u03bcM), and 6d/6a (10 \u03bcM) + L)\nF = 1\u2212 10\u2212 OD\n and further incubated for 4 h. The cells were stained with PI (60 \u03bcg/mL)\nwhere OD denotes the amount of light absorbed by the mixture at 520 and Calcein-AM (100 \u03bcg/mL) in PBS for 30 min. The fluorescence im-\nnm. ages were obtained immediately using a confocal microscope. Light\n\n 12\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\ndose: 520 nm, 200 mW/cm2, 10 min. RuB (10 \u03bcM), RuB (10 \u03bcM) + L, 6d (10 \u03bcM), and 6d (10 \u03bcM) + L) and\n further incubated for 24 h. Then the culture medium was removed, and\n4.12. Cellular co-localization assay the cells were lysed to collect the cell lysate. Subsequently, the con-\n centration of GSH content was measured using a GSH assay kit according\n Cells were seeded on confocal dishes at a density of 2 \u00d7 105 cells/mL to the manufacturer\u2019s instructions (Beyotime).\nin 2.0 mL culture medium and allowed to adhere overnight. Subse-\nquently, the cells were treated with 6d/6a (10 \u03bcM) for 4 h at 37 \u25e6 C in the 4.17. Western blot analysis\nabsence of light. Following this incubation period, the cells were washed\nwith phosphate-buffered saline. Next, the cells were further incubated Briefly, A549 cells were seeded in a six-well plate with fresh DMEM\nwith either LysoTracker Green (1 \u03bcM) or Mito-Tracker Green (75 nM) for containing 10 % FBS (2.0 mL per well) and incubation for 24 h.\n30 min at 37 \u25e6 C in the dark. The cells were washed three times with Following incubation, the cells were subjected to various treatments:\nphosphatebuffered saline to remove excess dye. Confocal images were (control, L, 6d (10 \u03bcM), and 6d (10 \u03bcM) + L) and further incubated for\nacquired using a confocal microscope to visualize the localization of the 24 h. After being washed twice with cold DPBS, the A549 cells were\ncomplexes within the cells. Mito-Tracker Green: \u03bbex = 460 nm, \u03bbem = treated with of ice-cold lysis buffer (60 \u03bcL). Upon disruption, the cell\n505\u2013525 nm; 6a: \u03bbex = 459 nm, \u03bbem = 600\u2013650 nm; 6d: \u03bbex = 460 nm, lysate was collected and boiled for 300 s at 95 \u25e6 C. Subsequently, the cell\n\u03bbem = 600\u2013650 nm. Lyso-tracker Green: \u03bbex = 460 nm, \u03bbem = 490\u2013520 lysates underwent SDS-polyacrylamide gel electrophoresis (SDS-PAGE)\nnm; 6a: \u03bbex = 459 nm, \u03bbem = 620\u2013670 nm; 6d: \u03bbex = 460 nm, \u03bbem = and a polyvinylidene fluoride (PVDF) membrane was used to transfer\n620\u2013670 nm. protein. The PVDF membrane was then blocked with skim milk (5 %\n TBST). Following this, the membrane was immunoblotted with a\n4.13. Intracellular ROS detection Glutathione Peroxidase 4 (GPX4) rabbit polyclonal antibody (dilution:\n 1:1000) and an anti-\u03b2-actin antibody (dilution: 1:2000) for 15 h. Sub-\n Intracellular generation of ROS was visualized using fluorescence sequently, the membrane was incubated with secondary antibody\nimaging with a DCFH-DA probe. Following a 24 h incubation and (dilution: 1:10,000) coupled with peroxidase (HRP) for another 1.5 h.\nremoval of the culture medium, the PBS group was set aside. Cells were Finally, specific protein bands were captured using an Omega Lum C\nthen treated with 6a/d (10 \u03bcM) and incubated for 4 h. After that, DCFH- Imaging System.\nDA (10 \u03bcM) was added as a ROS detection reagent, and the cells were\nincubated for another 30 min. The cells in the light group were subjected\n 4.18. Detection of lipid peroxidation\nto irradiation with a 520 nm laser (200 mW/cm2, 10 min). The fluo-\nrescence images were then obtained using a confocal microscope.\n Lipid peroxidation of 6d was visualized using fluorescence imaging\n using the C11-BODIPY 581/591 probe. Following a 24 h incubation and\n4.14. Mitochondrial membrane potential (MMP) detection\n removal of the culture medium, the cells were subjected to various\n treatments: (control, L, 6d/RuB (10 \u03bcM), and 6d/RuB (10 \u03bcM) + L) and\n Cells were seeded on confocal dishes at a density of 1 \u00d7 105 cells/mL\n further incubated for 4 h. Subsequently, the cells were washed with PBS\nin 2.0 mL culture medium and allowed to adhere upon incubation for 12\n and treated with C11-BODIPY (5 \u03bcM) for 30 min. Fluorescence imaging\nh. Following removal of the culture medium, the cells were subjected to\n was conducted using conventional filters: Texas Red (581/591 nm) and\nvarious treatments: (control, L, 6d/6a (10 \u03bcM), and 6d/6a (10 \u03bcM) + L)\n FITC (488/510 nm). Simultaneously, data acquisition for oxidized\nand further incubated for 4 h. Next, the cells were further incubated with\n BODIPY was efficiently carried out at its excitation maximum of 488 nm\nJC-1 solution (10 \u03bcg/mL) for 20 min at 37 \u25e6 C. The fluorescence images\n and emission maximum of 510 nm.\nwere obtained immediately using a confocal microscope. Light dose:\n520 nm, 200 mW/cm2, 10 min. Green channel: Ex, 488 nm; Em, 510\u2013545\nnm; Red channel: Ex: 535 nm; Em: 580\u2013610 nm. 4.19. Cell death mechanism\n\n\n4.15. Intracellular malondialdehyde (MDA) level detection To investigate the cell death mechanism, the cell viability was\n assessed following preincubation with various inhibitors targeting\n A549 cells were seeded in a six-well plate with fresh DMEM con- different cell death pathways. Cells were seeded in a 96 well plate at a\ntaining 10 % FBS (2.0 mL per well) and incubation for 24 h. Following density of 1 \u00d7 105 cells/mL in 2.0 mL culture medium and allowed to\nincubation, the cells were subjected to various treatments: (control, L, adhere upon incubation for 24 h. Preincubation with 3-Methyladenine\nRuB (10 \u03bcM), RuB (10 \u03bcM) + L, 6d (10 \u03bcM), 6d (10 \u03bcM) + L, 6d (10 \u03bcM) (100 \u03bcM), Ferrostatin-1 (10 \u03bcM), Necrostatin-1 (50 \u03bcM) or Z-VAD-\n+ Fer-1 (1 \u03bcM), and 6d (10 \u03bcM) + Fer-1 (1 \u03bcM) + L) and further incu- FMK (5 \u03bcM) were carried out for 60 min. Subsequently, 6d (10 \u03bcM) was\nbated for 24 h. Then the culture medium was removed, and the cells added and incubated for 4 h. Afterward, the cells were washed and\nwere lysed to collect the cell lysate. Subsequently, the concentration of subjected to irradiation (520 nm, 200 mW/cm2, 10 min). Cells in the\nMDA content was measured using a standard MDA assay kit. The non-irradiation group were replaced with fresh culture medium and\nabsorbance of the supernatants was measured using a multifunction kept in the dark. After an additional 20 h of co-incubation, with or\nmicroplate reader at wavelengths of 532 nm and 600 nm according to without the respective inhibitors, the survival rate of A549 cells was\nthe provided equation. determined using the MTT assay.\n\n\u0394OD = OD(Test) \u2212 OD(Blank) 4.20. Immunofluorescence staining of calreticulin (CRT)\n 4\nMDA (nmol/10 cell) = 0.1075 \u00d7 (\u0394OD532 nm \u2212 \u0394OD600 nm)\n Following incubation with 6d (10 \u03bcM) for 4 h, the supernatant was\nwhere OD denotes the absorbance values at the excitation wavelength. refreshed with fresh culture medium prior to irradiation. Following an\n additional 20 h incubation, cells were subjected to staining usin CRT\n4.16. Intracellular GSH level detection (D3E6) XP\u00ae Rabbit mAb, Anti-Rabbit IgG (H + L), F (ab\u2019)2 Fragment\n (Alexa Fluor\u00ae 647 Conjugate), and DAPI for 15 min following PBS\n A549 cells were seeded in a six-well plate with fresh DMEM con- washing. Subsequently, confocal microscopy was employed to image the\ntaining 10 % FBS (2.0 mL per well) and incubation for 48 h. Following slides, with excitation at 633 nm and emission collected at 690 \u00b1 40 nm.\nincubation, the cells were subjected to various treatments: (control, L, The specific operation is to refer to the method in the literature [50].\n\n 13\n\fH. Zheng et al. European Journal of Medicinal Chemistry 279 (2024) 116909\n\n\n4.21. Immunofluorescence staining of extracellular HMGB1 interests or personal relationships that could have appeared to influence\n the work reported in this paper.\n The cell treatment mirrored that of the CRT experiment. Instead of\nCRT (D3E6) XP\u00ae Rabbit mAb, HMGB1 Antibody was utilized. Confocal Data availability\nmicroscopy was employed to image the cells, with excitation at 514 nm\nand emission collected at 620 \u00b1 20 nm. Data will be made available on request.\n\n4.22. Extracellular ATP detection assay Acknowledgments\n\n The protocol for detecting extracellular ATP involved employing the This work was supported by the National Natural Science Foundation\nATP Bioluminescence Detection Kit (Promega). Post-collection, 100 \u03bcL of China (21977058 and 82473840); Key R&D Program of Jiangsu\nof supernatant was mixed with an equivalent volume of reagents in Province (BE2021677); China Postdoctoral Science Foundation\nopaque-walled plates. Subsequent quantification of ATP chem- (2018T110533); Key Natural Science Foundation of Jiangsu Higher\niluminescence signals was carried out using the TECAN Infinite M200 Education Institutions (20KJA350002); and Jiangsu Province Innova-\nPRO multifunctional reader. Correction of actual ATP content values tion Project of Postgraduate Training (KYCX22_3380 and\nacross groups was conducted to adjust for differences in cell viability KYCX24_3559).\nunder varied culture conditions.\n Appendix A. Supplementary data\n4.23. Tumor mice model\n Supplementary data to this article can be found online at https://doi.\n All animal experiments were conducted in accordance with the org/10.1016/j.ejmech.2024.116909.\nguidelines and regulations set forth by the Animal Research and Care\nCommittee of Nantong University (Approval No.: S20210925-003). For References\nthe establishment of the tumor-bearing mouse model, female BALB/c\nnude mice (5\u20136 week), with an average weight of around 20 g, were [1] A. Jassim, E.P. Rahrmann, B.D. Simons, R.J. Gilbertson, Cancers make their own\n luck: theories of cancer origins, Nat. Rev. Cancer 23 (2023) 710\u2013724.\nused. A suspension containing 5 \u00d7 105 A549 or 4T1 cells in PBS buffer [2] C. Thakur, F. 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