More-Is-Better Strategy for Constructing Homoligand Polypyridyl Ruthenium Complexes as Photosensitizers for Infrared Two-Photon Photodynamic Therapy.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n More-Is-Better Strategy for Constructing Homoligand Polypyridyl\n Ruthenium Complexes as Photosensitizers for Infrared Two-Photon\n Photodynamic Therapy\n Shi-Jie Tang, Meng-Fan Wang, Rong Yang, Meng Liu, Qing-Fang Li, and Feng Gao*\n Cite This: Inorg. Chem. 2023, 62, 8210\u22128218 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: Photodynamic therapy (PDT) uses a combination of\n photosensitizers (PSs), light sources, and reactive oxygen species (ROS)\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:17:11 (UTC).\n\n\n\n\n to damage only the desired target and keep normal tissues from being\n hurt. The dark cytotoxicity (chemotoxicity) of PSs, leading to whole-body\n damage in the absence of irradiation, is a major limiting factor in PDT.\n How to simultaneously increase ROS generation and decrease dark\n cytotoxicity is an essential challenge that must be resolved in PS research.\n In this study, a series of homoligand polypyridyl ruthenium complexes\n (HPRCs) containing three singlet oxygen (1O2)-generating ligands (L) in\n a single molecule ([Ru(L)3]2+) have been constructed. Compared to the\n heteroligand complexes [Ru(bpy)2(L)]2+ where bpy is 2,2\u2032-bipyridine, the\n 1\n O2 quantum yield under infrared two-photon irradiation and the DNA\n photocleavage effect of the HPRCs are significantly enhanced with two\n more ligands L. The intraligand triplet excited states transition played an important role in the activation of oxygen. The HPRCs\n target the mitochondria but not the nuclei, generating 1O2 intracellularly under irradiation of visible or infrared light. Ru1 exhibits\n high phototoxicity and low dark cytotoxicity toward human malignant melanoma cells in vitro. Moreover, HPRCs have minimal\n cytotoxicity to human normal liver cells, suggesting their potential as antitumor PDT reagents with more security. This study may\n provide inspiration for the structural design of potent PS for PDT.\n\n\n 1. INTRODUCTION Many Ru(II) complexes have been successfully developed as\n Surgical, chemotherapeutic, and radiation therapies are PSs in PDT and photochemotherapy (PCT) for their long 3ES\n commonly applied in cancer treatments. However, these lifetimes and high 1O2 quantum yields (\u03a6\u0394).7,8 Although some\n technics have certain disadvantages, including invasiveness, a good Ru(II)-PSs like TLD1433, which entered a clinical trial in\n high recurrence rate, substantial systemic toxicity, and side 2018, can be excited by red light (better than traditional blue\n effects.1,2 Photodynamic therapy (PDT) is an alternative to light), even longer excitation wavelength, especially the infrared\n chemotherapy, which mainly depends on a photosensitizer (IR) light, will be more practical for a deeper (typically >1 cm)\n (PS), oxygen molecules, and a light source. The PSs are and safer PDT. Ru(II), Ir(III), and Os(II) complexes with\n innoxious to cells or tissues without irradiation and only work in distinct IR two-photon absorption (TPA) have exhibited\n the tumor area exposing to light, thus have a low risk of side versatile properties in antitumor phototherapy.7,9\u221213 Currently,\n effects. The PS is first excited by light from the ground state (GS, the two-photon (TP) excitation depends on lasers with\n S0) to singlet excited states (1ESs) and subsequently relaxes from extremely high energy density. The power exceeds the maximum\n higher1ESs (Sn, n > 1) to S1 through internal conversion (IC) allowable exposure value (0.33 W cm\u22122) under IR laser for\n and transfers from1ESs to more stable triplet ESs (3ESs) by human skin (ANSI Z136.1-2014). In addition, the high-power\n intersystem crossing (ISC).2 The PS in 3ESs can produce ROS laser beam\u2019s restricted light spot size makes it hard to cover the\n by ES electron transfer/hydrogen atom abstraction (Type I) or solid tumors entirely, which severely limits its applicability and\n generate highly active singlet oxygen (1O2) by ES energy transfer\n (Type II). The light-generated ROS, notably 1O2, can react with Received: February 21, 2023\n biological targets such as lipids, proteins, and nucleic acids, Published: May 17, 2023\n destroy the targeted cells, damage the vasculature associated\n with the tumor, and stimulate antitumor immunity.3\u22126\n Consequently, the characteristic of 3ES and the ROS quantum\n yield are two crucial aspects of a PS.\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c00585\n 8210 Inorg. Chem. 2023, 62, 8210\u22128218\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nefficiency. As another option, the high-power xenon lamp with malignant melanoma cells under irradiation of visible (450 nm)\nhigh exposure usually causes damage to the tumor\u2019s surrounding and IR (808 nm) light and very low toxicity to human normal\ntissues due to its extensive irradiation area. liver cells. We anticipate that our study will spark interest among\n Recently, we designed a dinuclear Ru(II) complex12 with researchers in HPRC-type PSs and give meaningful strategies for\nremarkable TPA cross-section (TPACSs, \u03c3) and photothermal PS design in antitumor PDT.\nconversion efficiency (PCE) in the IR region and realized its\ndual PDT and photothermal therapy (PTT) in vivo by using a 2. EXPERIMENTAL SECTION\nlow-power laser (LPL, 808 nm, 100 mW cm\u22122) with a tunable 2.1. Materials. Ruthenium chloride (RuCl3), potassium hexafluor-\nbeam diameter (>1.0 cm) which has demonstrated good ophosphate (KPF6), ethylene glycol, N-ethyl-morpholine, neutral\napplicability in the antitumor phototherapy of upconverting alumina (200\u2212300 mesh), and organic solvents were purchased from\nnanoparticles.14 Exploring promising Ru(II)-PSs with simpler Adamas. Ammonium acetate, propanoic acid, aqueous ammonia,\nstructures and similar high phototherapy indexes (PIs) will CDCl3, and DMSO-d6 were purchased from Energy Chemical.\nundoubtedly be beneficial to pharmaceutical manufacturing. In a 2.2. Cell Lines and Culture Conditions. Human malignant\nprevious study, a ligand sip (Figure 1) has shown a high \u03a6\u0394 melanoma A375 and normal liver HL-7702 cell lines (STR\n authentication, Animal Research and Resource Center) were cultured\n in RPMI 1640 medium (Adamas Life) supplemented with 10% (v/v)\n FBS (Gibco BRL) at 37 \u00b0C in a carbon dioxide incubator (95% air and\n 5% CO2) with a humidified atmosphere. The tumor cells were routinely\n subcultured twice per week by trypsin\u2212EDTA treatment. The cells in\n an exponential growth phase were harvested and counted for tumor\n inoculation.\n 2.3. Subcellular Colocalization. A375 cells were grown on an 8-\n Chamber Glass Slide (Thermo Fisher Scientific) at a density of 6 \u00d7 104\n cell ml\u22121 and incubated for 1 h with the HPRCs at 2.5 \u03bcM. The cells\n were washed with PBS twice. Nuclei and mitochondria were\n counterstained with Hoechst 33342 (Invitrogen) and MitoTracker\n Green (MTG, Invitrogen), respectively. Luminescence images were\n collected on a Leica TCS SP8 DIVE two-photon confocal laser\n scanning microscopy (CLSM) at three detection channels (MTG: \u03bbex =\n 488 nm, \u03bbem = 510\u2212520 nm; HPRC: \u03bbex = 808 nm, \u03bbem = 610\u2212650 nm;\n Hoechst: \u03bbex = 405 nm, \u03bbem = 460\u2212500 nm) and processed by LAS X\n (Leica) software.\n 2.4. Cell Uptake. Exponentially growing A375 cells (2 \u00d7 107 cell\n ml\u22121) were harvested, and the resulting single-cell suspension was\n plated into 100 mm tissue culture plates (Adamas). After 24 h of\n incubation (37 \u00b0C, 5% CO2), the cells were incubated with 5 \u03bcM\n HPRC for 2 h at 37 \u00b0C in RPMI 1640 medium with 10% (v/v) FBS.\n The cells were rinsed with PBS, detached with trypsin, counted and\n divided into three portions: (1) in portion 1, the nuclei were extracted\n using a nucleus extraction kit (Thermo); (2) in portion 2, the cytoplasm\n was extracted using a cytoplasm extraction kit (Thermo); and (3) in\n portion 3, the mitochondria were extracted using a mitochondrial\n extraction kit (Thermo). All extraction procedures followed the\n manufacturer\u2019s protocols. To verify the HPRC concentration-depend-\nFigure 1. Homoligand polypyridyl ruthenium complexes Ru1\u2212Ru3 ent cell uptake, the same amount of A375 cells was incubated with 5, 50,\ndesigned in this work and previous heteroligand complex [Ru- and 200 \u03bcM of HPRC for 2 h at 37 \u00b0C in RPMI 1640 medium with 10%\n(bpy)2(sip)]2+(Ru0). (v/v) FBS and 5% DMSO. The samples were digested with 60% HNO3\n at room temperature for 1 day. Each sample was diluted with water to\n obtain 2% HNO3 sample solutions. The ruthenium content was\nvalue. The Ir(III) complex of sip has negligible DNA affinity and measured by a PlasmaQuant PQ9000 inductively coupled plasma mass\nvery low dark cytotoxicity and serves as a potent PS with high spectrometry (ICP-MS).\nPI.15 However, its Ru(II) analog Ru0 (Figure 1) is unlikely to be 2.5. Intracellular Singlet Oxygen Generation. 2\u2032,7\u2032-Dichlor-\na PS for PDT even though it shows remarkable photo- odihydrofluorescein diacetate (DCFH-DA, Thermo) was used as a\n fluorescent probe to detect the intracellularly generated ROS. A375\ncytotoxicity, due to its nonignorable chemotoxicity (dark cells were seeded in a cell culture dish. After 24 h of incubation (37 \u00b0C,\ncytotoxicity).16 5% CO2), the cells were incubated with HPRC solution (5 \u03bcM) in the\n In this study, three homoligand polypyridyl Ru(II) complexes absence and presence of ABDA (100 \u03bcM) for 2 h at 37 \u00b0C. After rinsing\n(HPRCs) containing three sip-type ligands in a single molecule with PBS, the cells were incubated with DCFH-DA (10 \u03bcM) for 20 min,\n([Ru(L)3]2+) have been constructed (Ru1\u2212Ru3, Figure 1). The washed with 3 \u00d7 1 mL PBS, irradiated with a 450 nm LED array (50\nlight-induced \u03a6\u0394 and the DNA photocleavage effect of HPRCs mW cm\u22122) or an 808 nm LPL (100 mW cm\u22122) for 5 min, and\nare anticipated to be significantly enhanced, compared with the photographed by CLSM as described above.\nparent complex Ru0 due to the increased number of highly 2.6. In Vitro Cell Viability (CCK-8 Assay). The cell viability was\nefficient 1O2-generating ligand sip. The DNA affinity of HPRC is determined by the CCK-8 assay. For cytotoxicity to A375 cells,\n exponentially grown cells were seeded in 384-well plates, followed by a\nexpected to reduce greatly as the steric hindrance of the two 24 h incubation for attachment. Cells were incubated with different\nexternal ligands became unignorable when one ligand attempts concentrations of HPRCs or cisplatin. For phototoxicity studies, after\nto insert itself into DNA. Their increased lipophilicity is found to 12 h of incubation, the supernatant was replaced with fresh culture\nbe beneficial for more efficient cell uptake. The HPRCs have medium and cells were subjected to irradiation by the 450 nm LED\nshown remarkable in vitro phototherapy indexes to human array (50 mW cm\u22122, light dose = 30.0 J cm\u22122) and 808 nm LPL (100\n\n 8211 https://doi.org/10.1021/acs.inorgchem.3c00585\n Inorg. Chem. 2023, 62, 8210\u22128218\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nmW cm\u22122, light dose = 30.0 J cm\u22122) and incubated for an additional 48\nh. Cells without irradiation were replaced with a fresh culture medium\nand maintained in the dark. Then, 10 \u03bcL of CCK-8 (Adamas Life)\nworking solution was added and incubated for 1 h. Absorbance at 450\nnm was measured on an iMark (Bio-rad) microplate reader, before and\nafter CCK-8 incubation. Data were reported as the mean \u00b1 standard\ndeviation (n = 3). IC50 values were determined by plotting the\npercentage of viability versus concentration on a logarithmic graph. For\ncytotoxicity to normal cells, exponentially grown HL-7702 cells were\nused instead.\n 2.7. Apoptosis Assay by Flow Cytometry Analysis. Cell\napoptosis was measured by Annexin V-FITC (fluorescein isothiocya-\nnate, AV) and propidium iodide (PI) based on the manufacturer\u2019s\nmanual (Beyotime). Human melanoma cells A375 at a density of 1 \u00d7\n105 cell mL\u22121 in 1 mL of RPMI 1640 medium containing 10% FBS and\n1 \u00d7 103 units penicillin/streptomycin were incubated with 1 \u03bcM of\nHPRC at 37 \u00b0C (5% CO2) for 2 h. The HPRC-containing medium was\nreplaced with a fresh HPRC-free medium. Cells were irradiated by the Figure 2. Absorption (solid) and emission spectra (dashed, \u03bbex = 455\n450 nm LED array (50 mW cm\u22122, light dose = 30.0 J cm\u22122), 808 nm nm) of Ru1\u2212Ru3 (10 \u03bcM) in anhydrous acetonitrile at 20 \u00b0C.\nLPL (100 mW cm\u22122, light dose = 30.0 J cm\u22122) or away from the light for\n10 min at room temperature, stained with AV/PI or AV alone, and\nimmediately examined with a BD FACSCalibur flow cytometer via two Table 1. Spectroscopy and Electrochemistry Data for HPRCs\nchannels or single channel. Ru1\u2212Ru3\n 2.8. Caspase Activation. Caspase-3 and -9 activities were HPRC \u03bbabs/nm (\u03b5/\u00d7104 M\u22121 cm\u22121) \u03bbem/nm \u03a6/% Eox/V Ered/V\nmeasured through cleavage of a colorless substrate specific for\ncaspase-3 (Ac-DEVD-pNA) or caspase-9 (Ac-LEHD-pNA) releasing Ru1 471 (2.68), 437 (2.08), 348 603 0.63 0.71 \u22121.38,\n (8.80), 296 (10.24) \u22121.56,\np-nitroaniline (pNA). A375 cells were seeded in 96-well plates (1 \u00d7 105 \u22122.06\ncells/well) and allowed to incubate for 6 h. The cells were then Ru2 474 (2.23), 393 (8.20), 282 626 2.8 0.72 \u22121.31,\nincubated respectively with control (PBS) or HPRC in the dark. After (9.68) \u22121.52,\n12 h incubation, the cells were divided into two groups. The dark \u22122.09\ngroups were incubated for an additional 12 h, and the irradiation groups Ru3 472 (2.40), 441 (1.93), 359 600 0.41 0.65 \u22121.39,\nwere exposed to 808 nm LPL (100 mW cm\u22122, light dose = 30.0 J cm\u22122) (8.20), 298 (7.43) \u22121.50,\n \u22122.08\nbefore they were incubated for 12 h in the dark. The cells were lysed and\ntreated with a caspase-3 or -9 activity kit (Beyotime) according to the\nmanufacturer\u2019s protocol. Absorbance at 405 nm was measured on an The emission spectra of Ru1\u2212Ru3 exhibited typical\niMark (Bio-rad) microplate reader. The caspase-3 and -9 activities in characteristics for Ru(II) complexes (Figure 2), with emission\ndrug-treated cells were determined as relative values to control groups band maxima focused around 610 nm (Table 1). From the\n(PBS). Data were reported as the mean \u00b1 standard deviation (n = 3). quantum yields of emission (\u03a6) in anhydrous MeCN, the\n emission intensity of Ru2 (\u03a6 = 0.028) is quite moderate,\n3. RESULTS AND DISCUSSION compared with the reference [Ru(bpy)3]2+ (\u03a6 = 0.095 in\n 3.1. Synthesis and Photostability. The synthesis of Ru1\u2212 MeCN). Ru1 and Ru3 emit even fainter. In an aqueous solution,\nRu3 and their characterization, including 1H NMR (Figure S1), all HPRCs are nearly nonemitting (<5 \u03bcM) through one-\n13 photon1MLCT excitation due to the quenching of water\n C NMR, ESI-MS, and microanalysis, are shown in the\n molecules. Theoretical analysis revealed that the emission of\nSupporting Information. In comparison to Cl-coordinated HPRCs arose from the intraligand (3IL) transition (Figures 3b,c,\nRu(II)-arene complexes, coordination-saturated PRCs are S5, and S6). A low-energy 3IL state is suggested to be a\nknown to be inert to ligand substitution.17,18 By examining the significant factor for the 1O2 production of a transition metal-\nabsorption spectral changes of Ru(II) complexes in the absence based PS, such as TLD1433.8 Therefore, the above studies on\nof light, no hydrolysis was observed for Ru1\u2212Ru3 in the the ES properties of HPRCs elucidated plausible implications\naqueous solution (Tris\u2212HCl buffer, Figure S2) or RPMI 1640 for their good performances in PDT.\ncell culture medium (Figure S3). Upon light irradiation with a 3.3. Electrochemistry. The GS oxidative and reductive\n450 nm LED (50 mW cm\u22122) or an 808 nm LPL (100 mW potentials (Eox and Ered) of Ru1\u2212Ru3 were studied in degassed\ncm\u22122), Ru1\u2212Ru3 are also stable, as the spectra showed nearly anhydrous CH3CN by CV and summarized in Table 1. Ru1\u2212\nno change in the MLCT bands. Ru3 exhibited irreversible couples ranging from 0.65 to 0.72 V,\n 3.2. Spectroscopy and DFT Calculations. The absorption which were attributed to Ru(III)/Ru(II) oxidation.21,22 Due to\nspectra of Ru1\u2212Ru3 (10 \u03bcM) were recorded in the acetonitrile the presence of the electron-withdrawing \u2212NO2 group, the\nsolution (Figure 2), and the absorption band maxima (\u03bbabs) and electron-accepting ability of Ru2 is enhanced, and its oxidation\nmolar extinction coefficients (\u03b5) are listed in Table 1. In the potential is shifted toward the anode compared to Ru1. The\nrange of 400\u2212480 nm, absorption bands were attributed to the oxidation potential of Ru3 moves to the cathode due primarily\nmetal-to-ligand charge transfer (MLCT, dRu to \u03c0L*) transi- to the function of electron-donating groups. Three reduction\ntions,19,20 as shown in the simulated absorption spectra (Figures couples (\u22121.31 to \u22121.39, \u22121.50 to \u22121.57, and \u22122.06 to \u22122.09\n3a and S4) by TDDFT, the simplified Jablonski diagraph V) representing the reduction with sip as the core were observed\n(Figure 3b) and the real space representation of hole and for Ru1\u2212Ru3, and the redox potentials are comparable to those\nelectron distributions (Figures 3c, S5, and S6). Bands between of previously reported sip-containing complexes.\n350 and 400 nm are attributed to the ligand-centered (LC, \u03c0L to 3.4. Singlet Oxygen Quantum Yield. The 1O2 quantum\n\u03c0L*) transitions, as in Ru0 and its analogs.16 yields (\u03a6\u0394, Table 2) for each HPRC and Ru0, as well as\n 8212 https://doi.org/10.1021/acs.inorgchem.3c00585\n Inorg. Chem. 2023, 62, 8210\u22128218\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n [Ru(bpy)3]2+ (Ref), were determined by the absorbance\n variations of ABDA at 378 nm (Figures 4, and S7\u2212S9). Upon\n irradiation of 450 nm LED (50 mW cm\u22122), the \u03a6\u0394450 values for\n Ru1 and Ru3 were 0.59 and 0.49, respectively, suggesting a high\n 1\n O2 generation efficiency. Compared to Ru0 (0.13), the \u03a6\u0394450 of\n Ru1 increased approximately fourfold, showing that substituting\n the two bpy ligands with sip can significantly boost the 1O2\n production of the Ru(II) complex, which has a substantial effect\n on the efficacy of PDT. Under 808 nm LPL (100 mW cm\u22122)\n irradiation, Ru1 also has the highest \u03a6\u0394808 value among these\n complexes. This can be rationalized from two aspects. On the\n one hand, as shown in the TDDFT calculations and real space\n distributions of ES (Figures S2\u2212S4), all the lowest three 3ESs are\n 3\n IL transitions for each HPRC, which support a high PDT\n capacity.23,24 On the other hand, Ru1 exhibited a lower 3IL\n energy level than Ru2 and Ru3 (Figures 3c, S2, and S3),\n implying a more efficient intersystem crossing (ISC) pathway to\n reach the 3ES which is responsible for energy transfer in type II\n PDT.25 The high 1O2 quantum yield of Ru1\u2212Ru3 may lead to\n enhanced DNA photocleavage and phototoxicity. Since no IR\n absorption of the HPRCs was observed in the absorption\n spectra, the origin of good exciting efficiency and 1O2 production\n of the HPRCs have been hypothesized to be the two-photon\n absorption property.\n 3.5. Two-Photon Absorption. TPACSs of HPRCs at 808\n nm (\u03c3808) have been measured by the well-established method\n involving two-photon-induced fluorescence.26 \u03c3808 of HPRCs\n was 51\u2212142 GM (1 GM = 10\u221250 cm4 s photon\u22121) as shown in\n Table 2, which was not only higher than the suggested value for\n optical imaging applications (0.1 GM)27 but also higher than\n similar mononuclear Ru(II)28,29 and Ir(III)30 complexes at\n around 800 nm. Nevertheless, the \u03c3808 values of HPRCs are\n lower than some dinuclear Ru(II),12 Os(II),13 and Ir(III)26\n complexes. Ru1 showed the highest \u03c3808, which could explain its\n remarkable photodynamic activity (\u03a6\u0394808) under 808 nm\n excitation.\n 3.6. Determination of n-Octanol and Water Distribu-\n tion Coefficient. Investigation into the lipophilicity provides\n an important basis for cell membrane penetration, cellular\n uptake and bioavailability of drugs and reported polypyridyl\n Ru(II) complexes.31\u221233 The distributions of Ru1\u2212Ru3 in n-\n octanol/water solutions were shown in Figure S10. The oil/\n water partition coefficients (log KO/W) were determined by the\n concentrations of complexes in both phases. It is evident that\n these complexes have optimum lipophilic properties (log KO/W\n = 1.47\u22122.34), which would make them not only enter cells\n efficiently but also have significant benefits in the process of\n pharmaceutical application, such as balanced volume of\n distribution and potential for good absorption and bioavail-\n ability.\n 3.7. Mitochondrial Colocalization. The subcellular target\n is crucial for the activity and mechanism of drugs.34 The\n subcellular colocalization of Ru1\u2212Ru3 with the mitochondria\n dye MitoTracker Green (MTG) was explored by CLSM on\n A375 human melanoma cells. All HPRCs showed weak\n luminescence within the cells under two-photon excitation at\nFigure 3. Electron transition analysis of Ru1 by quantum chemistry 808 nm (Figure 5a), which almost coincided with the signals of\ntheoretical calculations (B3LYP/Lanl2DZ, CPCM = water). (a)\n MTG but overlapped poorly with the nuclei dye Hoechst33324.\nExperimental and calculated absorption spectra of Ru1. (b) Simplified\nJablonski diagraph showing the excitation, emission, and energy The high Pearson\u2019s correlation coefficients (R) for the\ntransfer of Ru1. (c) Hole (blue) and electron (green) distributions of colocalization between MTG and HPRCs indicate that the\nRu1 for the excited singlet (1ES) and triplet states (3ES). Excitation complexes preferentially accumulate in the mitochondria. Their\nwavelength (nm), excitation energy (eV), and oscillator strength (f) for impermeable nature to the nucleus membrane or their low\neach transition are included. affinities for nuclear DNA could be factors impeding their\n 8213 https://doi.org/10.1021/acs.inorgchem.3c00585\n Inorg. Chem. 2023, 62, 8210\u22128218\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 2. Singlet Oxygen Quantum Yield at 450 nm (\u03a6\u0394450) and 808 nm (\u03a6\u0394808), Two-Photon Absorption Cross-Section at 808\nnm (\u03c3808, GM) In Vitro (Photo)Cytotoxicity (IC50, \u03bcM) and Phototherapy Index (PI) of Ru1\u2212Ru3 toward Thick-Bedded A375\nHuman Melanoma Cell Lines\n complex \u03a6\u0394450 \u03a6\u0394808 \u03c3808 IC50, dark IC50, 450 PI-1 IC50, 808 PI-2\n Ru1 0.59 0.11 142 >200 0.726 \u00b1 0.070 >275 0.513 \u00b1 0.042 >390\n Ru2 0.13 0.04 51 >200 53.6 \u00b1 4.8 >3.73 34.1 \u00b1 2.9 >5.87\n Ru3 0.49 0.08 97 >200 2.75 \u00b1 0.26 >72.7 2.51 \u00b1 0.18 >79.7\n Ru0 0.13 0.05 91 28.6 \u00b1 2.4 5.36 \u00b1 0.48 5.33 4.65 \u00b1 0.37 6.15\n\n 3.9. Intracellular 1O2 Generation. Given that the HPRCs\n possess efficient cell uptake and high 1O2 yields in the aqueous\n solution upon both VIS and IR light irradiations, their\n intracellular 1O2 generation abilities have been studied (Figure\n 5c) by the well-established DCFH-DA method. After either 450\n or 808 nm irradiation, all HPRCs induced bright luminescence\n of DCF over the entire cell, demonstrating the intracellular\n production of ROS. In the presence of 1O2 scavenger ABDA, no\n ROS generated in the complex-treated A375 cells under the\n same TP irradiation (Figure S11), indicating 1O2 is the main\n species in the photogenerated ROS by the HPRCs. Not\n surprisingly, Ru1 exhibited the most potent intracellular 1O2\n production, as it did in an aqueous solution. This can provide\n substantial support for the HPRCs to become effective PS for\n antitumor PDT.\n 3.10. Cytotoxicity to Cancerous Cells. The cytotoxicities\n of HPRCs Ru1\u2212Ru3 and Ru0 on the human melanoma cells\n A375 were evaluated by CCK-8 assay (Table 2). Under the dark\n condition, Ru1\u2212Ru3 showed negligible cytotoxicity at a\n concentration of 200 \u03bcM. Their half-maximal inhibitory\n concentrations in the dark (IC50, dark) were expressed as >200\n \u03bcM. This dark toxicity is apparently lower than most recently\n reported PSs based on transition metal complexes, such as\n hydrolysable [RuL2Cl2]-type complexes (<5 \u03bcM),4 cyclo-\n metalated Pt(II) complexes (\u223c17 \u03bcM),38 self-assembly arene-\n Ru(II) complexes,39 bis-terpyridyl Ru(II) complexes,40 and is\n similar to Ir-bodipy complexes,41 Ru-bodipy complexes,42 and\n HPRCs [Ru(dppz-X2)3]2+ based on classic dppz-type ligand\n (>100 \u03bcM).43 Upon irradiation with the 450 nm LED array and\n 808 nm LPL, the HPRCs, especially Ru1, induced remarkable\n cell death (IC50, 450 = 0.726 \u00b1 0.070 \u03bcM and IC50, 808 = 0.513 \u00b1\nFigure 4. Measurement of singlet oxygen quantum yield of HPRCs by\n 0.042 \u03bcM). The phototherapy indexes under 450 (PI-1) and\nABDA in aqueous solution upon 450 nm LED (50 mW cm\u22122) and 808 808 nm (PI-2) for Ru1 reached as high as >275 and > 390,\nnm LPL (100 mW cm\u22122) irradiation. respectively, indicating that it is an IR-excitable PS for antitumor\n PDT. Both PI-1 and PI-2 of HPRCs are significantly high among\neffective accumulation in the nuclei. As a result, mitochondria metal-containing PSs summarized in recent reviews (typically\nbecome the primary target of Ru1\u2212Ru3, consistent with some <100).3,5,6,44\u221249 The phototoxicity of HPRCs [Ru(dppz-X2)3]2+\nRu(II) complexes which were reported to be mitochondria- have been reported to decrease as their lipophilicity increases.43\ntargeted and challenging to enter the nucleus.35,36 Quite the contrary, for HPRCs in this work, a decrease in\n 3.8. Cell Uptake. Cell uptake and intracellular distribution lipophilicity leads to an apparent reduction in phototoxicity. The\nof Ru1\u2212Ru3 was further tested by ICP-MS (Figure 5b). The cell high activity of Ru1 is attributed to its enhanced cell uptake,\nuptake amounts of HPRCs are very comparable to typical metal brought about by its higher lipophilicity, and its greater\ncomplexes.37 The cytoplasm and nucleus contain 96.2 and 3.1% photoexcited 1O2 yield. The disparity in the structure\u2212activity\nof the whole cell\u2019s ruthenium, respectively, while 92.3% of the relationship of various series of HPRCs underlines a demand for\nruthenium in the whole cell has been found in the more broad and in-depth research.\nmitochondrion. The uptake of HPRC by A375 cells exhibited In stark contrast to HPRC, Ru0 showed moderate toxicity to\nclear concentration dependence (Figure S12). This further A375 cells without irradiation, similar to previously observed in\ndemonstrates the mitochondrial selectivity of HPRCs and their other tumor cell lines,16 and the cytotoxicity was less improved\neffective cell uptake capacity. The amount of HPRCs taken up under 450 or 808 nm irradiation. It demonstrated that replacing\nby cells also showed a positive correlation with their hydro- the auxiliary ligands bpy in Ru0 with sip-type ligands reduced\nphobicity (Ru1 > Ru3 > Ru2). Therefore, the optimized the dark toxicity (chemotherapy toxicity) and increased the\nlipophilicity of Ru1\u2212Ru3 is beneficial to their efficient cell photocytotoxicity. Ru0 has very high DNA affinity via\nuptake.33 intercalation mode, while the HPRCs are not potent DNA\n 8214 https://doi.org/10.1021/acs.inorgchem.3c00585\n Inorg. Chem. 2023, 62, 8210\u22128218\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n 3.11. Apoptosis Assay. Flow cytometry detection of\n apoptosis in A375 cells, stained with Annexin V-FITC (AV)\n and PI (Figures 6a and S16), has been further carried out for the\n\n\n\n\n Figure 6. Photoinduced A375 cell apoptosis and caspase activation by\n Ru1\u2212Ru3. (a) Flow cytometry detection of apoptosis in A375 cells\n (stained with Annexin V-FITC and PI) for the PDT treatments of\n HPRCs (1 \u03bcM) in the dark or upon 450 nm LED (50 mW cm\u22122) and\n 808 nm LPL (100 mW cm\u22122) irradiation. (b) Caspase-9 and -3\n activities in A375 cells in the presence of HPRC (1 \u03bcM) in the dark or\n upon 808 nm LPL irradiation (100 mW cm\u22122).\n\n PDT treatments of Ru1\u2212Ru3. The proportions of necrotic cells\n (Q1) were ignorable during the assay. In the absence of light,\n less than 2% of the HPRC-treated cells were found in the\n apoptotic stage (Q2), which is close to the number of cells in the\n blank group (A375 cells alone). It indicated that the HPRCs\n have minimal apoptosis-inducing activity in the dark. Upon light\nFigure 5. Colocalization of HPRCs with mitochondria (stained by irradiation, the apoptotic rates of the HPRC-treated cells\nMTG) in A375 cells by CLSM for Ru1\u2212Ru3 (a) and Ru distribution increased, especially for Ru1. Therefore, the remarkable in vitro\nfor Ru1\u2212Ru3 in different parts of the cell by ICP-MS (b), as well as phototherapy efficacy of Ru1 is primarily a result of the\nDCFH-DA detection of intracellular 1O2 generation for Ru1\u2212Ru3 (5 apoptotic pathway induced by light-generated singlet oxygen.\n\u03bcM) upon 450 nm LED (50 mW cm\u22122) and 808 nm LPL (100 mW HPRC-treated A375 cells stained by AV alone were also\ncm\u22122) irradiation for 5 min (c). Nuclei were stained by Hoechst 33324\n analyzed to confirm whether the flow cytometry results were\n(Hoechst). Pearson\u2019s correlation coefficients (R) were calculated for\nthe colocalization between MTG and HPRCs. The percentages affected by the emission of HPRCs as their emission maximums\nrepresent the relative contents of Ru in each examined part of the were very close to PI. The percentages of apoptotic cells\nwhole cell. represented by the AV signals (Figure S17a), both in the dark\n and upon irradiation, were almost identical to the total apoptosis\n rates indicated in the AV/PI analysis (Figure 6a). An additional\nbinders as revealed by the slight changes in absorption spectral PI channel detection of A375 cells treated by HPRC alone\ntitration (Figure S13) and the small DNA melting point (without PI in the dark) and stained by PI alone was also\nincreases in DNA denaturation (Figure S14). The possible performed (Figure S17b). The results indicated that the\nfactors may be the steric hindrance of large auxiliary ligands. The fluorescence of HPRC-treated cells had only a slight influence\nenhanced 1O2 quantum yields by introducing two more sip-type on the PI signals for the judgment of cell apoptosis. Although\nligands greatly boost their DNA photocleavage activities (Figure HPRCs emit in anhydrous organic solvents, their aqueous\nS15) and photocytotoxicities. solutions were almost non-emissive due to the quenching effect\n 8215 https://doi.org/10.1021/acs.inorgchem.3c00585\n Inorg. Chem. 2023, 62, 8210\u22128218\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nof water molecules (Figure S18). Based on the protection of yield determination for the 1O2 generation, DNA\nDNA base pairs, classic DNA-intercalative Ru(II) complexes photocleavage, oil/water partition, experimental and\nand organic dyes can recover their emission and serve as DNA computational absorption spectral data of HPRCs\nlight switches. For HPRCs, their emissions after binding to DNA (PDF)\nwere nearly the same as free HPRCs (Figure S18), suggesting\nthat they were still quenchable by water molecules, most\nprobably via the two sip ligands outside. Therefore, the signals\ndetected in flow cytometry under the concentrations in this\n \u25a0 AUTHOR INFORMATION\n Corresponding Author\nstudy almost entirely came from the DNA-bound PI and were Feng Gao \u2212 Key Laboratory of Medicinal Chemistry for Natural\nnot interfered with by the Ru(II) complexes. Resource, Ministry of Education; Yunnan Provincial Center for\n In the apoptotic process, caspase-9 propagates a cascade of Research & Development of Natural Products; School of\nfurther caspase processing events by directly cleaving and Pharmacy, Yunnan University, Kunming 650500, P. R.\nactivating caspase-3 and caspase-7 to execute cell death. China; orcid.org/0000-0001-7490-4887;\nCaspase-9 and -3 activities were measured in the HPRC-treated Email: gaofeng@ynu.edu.cn\nA375 cells (Figure 6b). All complexes exhibited no caspase-9 or Authors\n-3 activation under dark conditions. With 808 nm LPL Shi-Jie Tang \u2212 Key Laboratory of Medicinal Chemistry for\nirradiation, both caspase-9 and -3 activities rose substantially. Natural Resource, Ministry of Education; Yunnan Provincial\nThese findings demonstrate that the HPRCs initiate a photo- Center for Research & Development of Natural Products;\ndepended apoptosis mechanism. School of Pharmacy, Yunnan University, Kunming 650500, P.\n 3.12. Cytotoxicity to Noncancerous Cells. The toxicity of R. China\nPS to normal cells is one of the most commonly observed Meng-Fan Wang \u2212 Key Laboratory of Medicinal Chemistry for\nundesired effects of PDT in clinical settings. The cytotoxicities Natural Resource, Ministry of Education; Yunnan Provincial\nof HPRCs Ru1\u2212Ru3 and Ru0 to the human normal liver (HL- Center for Research & Development of Natural Products;\n7702) cells were tested using cell viability incubated with School of Pharmacy, Yunnan University, Kunming 650500, P.\ndifferent concentrations of Ru(II) complexes (Figure S18). Ru0 R. China\nshowed low toxicity to HL-7702 cells. At a concentration around Rong Yang \u2212 Key Laboratory of Medicinal Chemistry for\nthe IC50 for phototherapy in A375 cells (5.0 \u03bcM), Ru0 reduced Natural Resource, Ministry of Education; Yunnan Provincial\nHL-7702 cell viability by less than 10%. Despite a 10-fold Center for Research & Development of Natural Products;\nincrease in Ru0 concentration (50 \u03bcM), the HL-7702 cell School of Pharmacy, Yunnan University, Kunming 650500, P.\nviability was still 67%. The toxicities of Ru1\u2212Ru3 to normal cells R. China\nwere even lower than that of Ru0. For Ru1, the HL-7702 cell Meng Liu \u2212 Key Laboratory of Medicinal Chemistry for Natural\nviability was higher than 95% when the concentration reached Resource, Ministry of Education; Yunnan Provincial Center for\n5.0 \u03bcM (10 times higher than its phototherapeutic IC50). The Research & Development of Natural Products; School of\nlow cytotoxicity of HPRCs to normal cells greatly increased their Pharmacy, Yunnan University, Kunming 650500, P. R. China\npotential as high-selective and safe PSs for antitumor PDT. Qing-Fang Li \u2212 Key Laboratory of Medicinal Chemistry for\n Natural Resource, Ministry of Education; Yunnan Provincial\n4. CONCLUSIONS Center for Research & Development of Natural Products;\nIn summary, a series of homoligand polypyridyl ruthenium School of Pharmacy, Yunnan University, Kunming 650500, P.\ncomplexes ([Ru(L)3]2+) containing three singlet oxygen- R. China\ngenerating ligands (L) in a single molecule have been Complete contact information is available at:\nconstructed. The complexes exhibited significantly enhanced https://pubs.acs.org/10.1021/acs.inorgchem.3c00585\n1\n O2 quantum yield compared to their heteroligand parents,\nunder the irradiation of both visible light (450 nm) and infrared Notes\nlight (808 nm) by one- and two-photon absorption, respectively. The authors declare no competing financial interest.\nThe HPRCs target the mitochondria but not the nuclei. Ru1 has\na remarkable phototherapy index for human malignant\nmelanoma cells upon irradiation of 808 nm low-power laser\n \u25a0 ACKNOWLEDGMENTS\n This work was supported by the National Natural Science\nand very low toxicity to human normal liver cells, suggesting its Foundation of China (22167022), Yunnan Provincial Science\nhigh potential as IR-excitable antitumor PDT reagent with and Technology Department (2018FB022), Youth Talents\nimproved therapeutic efficacy and safety. We expect that our Project of Yunnan Province (QNBJ-2018-057). The authors\nstudy on the two-photon antitumor PDT activity of HPRC will thank the Advanced Analysis and Measurement Center of\npique the attention of those working in chemistry, pharmacy, Yunnan University for their help in characterization.\noptical materials, and other relevant domains in homoligand\nmetal complexes.\n \u25a0 REFERENCES\n\n\u25a0\n*\n ASSOCIATED CONTENT\ns\u0131 Supporting Information\n (1) Roque, J. A., III; Cole, H. 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