Bleeding the Excited State Energy to the Utmost: Single-Molecule Iridium Complexes for In Vivo Dual Photodynamic and Photothermal Therapy by an Infrared Low-Power Laser.

{"full_text": "RESEARCH ARTICLE\n www.advhealthmat.de\n\n\nBleeding the Excited State Energy to the Utmost:\nSingle-Molecule Iridium Complexes for In Vivo Dual\nPhotodynamic and Photothermal Therapy by an Infrared\nLow-Power Laser\nShi-Jie Tang, Qing-Fang Li, Meng-Fan Wang, Rong Yang, Li-Zhen Zeng, Xue-Lian Li,\nRui-Dong Wang, Hongbin Zhang, Xiaoxia Ren, Dan Zhang, and Feng Gao*\n\n 1. Introduction\nA series of cyclometalated Ir(III) complexes with morpholine and piperazine\ngroups are designed as dual photosensitizers and photothermal agents for Photodynamic therapy (PDT) is a promis-\n ing cancer treatment that involves the gen-\nmore e\ufb03cient antitumor phototherapy via infrared low-power laser. Their\n eration of reactive oxygen species (ROS) by\nground and excited state properties, as well as the structural e\ufb00ect on their photosensitizers (PSs) upon light irradia-\nphotophysical and biological properties, are investigated by spectroscopic, tion, resulting in noninvasive tumor cell\nelectrochemical, and quantum chemical theoretical calculations. They target death.[1] PDT is partially dependent on oxy-\nmitochondria in human melanoma tumor cells and trigger apoptosis related gen availability. Tumors, particularly big\nto mitochondrial dysfunction upon irradiation. The Ir(III) complexes, solid tumors, have a hypoxic environment\n that signi\ufb01cantly limits the e\ufb03cacy of PDT\nparticularly Ir6, demonstrate high phototherapy indexes to melanoma tumor\n due to rapid cell proliferation, limited vas-\ncells and a manifest photothermal e\ufb00ect. Ir6, with minimal cular development, and uneven distribu-\nhepato-/nephrotoxicity in vitro, signi\ufb01cantly inhibits the growth of melanoma tion. The combination of PDT with one\ntumors in vivo under 808 nm laser irradiation by dual photodynamic therapy or more other novel therapeutics, such as\nand photothermal therapy and can be e\ufb03ciently eliminated from the body. photothermal therapy (PTT), photoacoustic\n therapy (PAT), photochemotherapy (PCT),\nThese results may contribute to the development of highly e\ufb03cient\n and immunogenic cell death (ICD), can\nphototherapeutic drugs for large, deeply buried solid tumors. integrate their respective bene\ufb01ts, signi\ufb01-\n cantly improve treatment e\ufb03cacy, and re-\n duce the dosage of therapeutic agents.[2]\n Oxygen-independent PTT can be simply performed in con-\n junction with PDT, inducing thermal ablation of tumors without\n the need for additional equipment or reagents.[3] Neverthe-\n less, PSs based on monomolecular metal complexes, such as\nS.-J. Tang, Q.-F. Li, M.-F. Wang, R. Yang, L.-Z. Zeng, X.-L. Li, R.-D. Wang,\nH. Zhang, F. Gao [Ru(bpy)3 ]2+ and [Ir(ppy)2 bpy]2+ , have virtually no photothermal\nKey Laboratory of Medicinal Chemistry for Natural Resource conversion (PTC) capability. Therefore, the PTC e\ufb00ect is not\nMinistry of Education an inherent feature of metal complexes, and speci\ufb01c structural\nYunnan Provincial Center for Research & Development of Natural modi\ufb01cations are required for the development of photothermal\nProducts\nSchool of Pharmacy agents (PTAs) based on metal complexes. The reported metal\nYunnan University complexes that can be employed for dual PDT/PTT therapy com-\nKunming 650500, P. R. China prise ligands with aggregation tendencies and are preprepared in\nE-mail: gaofeng@ynu.edu.cn aggregation states, such as micelles, solid nanoparticles (NPs),\nX. Ren and so on.[4] Despite existing concerns regarding the safety of\nAnimal Research and Resource Center\n NPs, the manufacture of such complex aggregates, the variety\nSchool of Life Sciences\nYunnan University of materials employed, and the size heterogeneity of individual\nKunming 650091, P. R. China particles make their future pharmaceutical work extremely\nD. Zhang challenging. The encapsulation of multiple ingredients in the\nFirst A\ufb03liated Hospital of Kunming Medical University carrier or their conjugation with the carrier generally causes\nKunming 650032, P. R. China an excess of parenteral excipients and low active-ingredient\n loading. Therefore, it is really necessary to have a single ac-\n The ORCID identi\ufb01cation number(s) for the author(s) of this article tive component, which can eliminate the tedious procedures\n can be found under https://doi.org/10.1002/adhm.202301227 as well as improve the bioavailability of synergistic therapy\nDOI: 10.1002/adhm.202301227 agents.[3,5]\n\n\nAdv. Healthcare Mater. 2023, 12, 2301227 2301227 (1 of 9) \u00a9 2023 Wiley-VCH GmbH\n\f 21922659, 2023, 28, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202301227 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 1. Structures and characteristics of cyclometalated Ir(III) complexes Ir1\u20136 designed in this study as IR two-photon excitable single-molecule\nphotosensitizers/photothermal agents (PSs/PTAs) for in vivo antitumor photodynamic/photothermal therapy (PDT/PTT).\n\n\n\n To this end, we have realized the single-molecule antitumor the bodies of mice within 14 d, with just trace amounts remain-\ndinuclear Ru(II) complexes for dual PDT/PTT in vivo by mod- ing in the liver, kidneys, and tumors. Further research revealed\nulating triple excited states (3 ESs) with a lowered energy level that Ir6 has negligible hepatorenal toxicity without IR irradiation,\nfor e\ufb03cient energy transfer to oxygen and a large rotatable sub- increasing con\ufb01dence in its safety for further pharmacological\nstituent group for convenient nonradiative (exothermic) decay.[6] study.\nIntriguingly, their dinuclear Os(II) counterparts have 3 ESs with\neven lower energy, rendering practically all of their ES energy 2. Results and Discussion\ndecaying via heat release, resulting in an enhancement in PTT\nactivity and an elimination of PDT activity.[7] The balance of ES 2.1. Photophysical Properties\nenergy in three aspects, namely, radiation decay (luminescence),\nnonradiation decay (heat release), and energy transfer with oxy- The UV\u2013vis absorption spectra of Ir1\u20136 in aqueous solution at\ngen, may be crucial to the therapeutic mechanism and e\ufb03cacy of 25 \u00b0C were recorded (Figure 2a). The band maxima (\ud835\udf06abs ) and\na PS or/and PTA. molar extinction coe\ufb03cients (\ud835\udf16) were summarized in Table 1.\n Cyclometalated Ir(III) complexes are widely used as lumines- The absorbance follows the Beer\u2013Lambert law in the concentra-\ncent materials and antitumor PSs due to their extended 3 ES tion range of 1\u201350 \u00d7 10\u22126 m, suggesting the absence of ground\nlifetime and high quantum yield of both emission and 1 O2 state aggregation in this tested concentration range. The spectra\nproduction.[8] In this study, by introducing vibrational hetero- of Ir1\u20136 are comprised of three main absorption bands. With the\ncyclic groups onto a pip-type ligand and rotatable methyl sub- assistants of time-dependent density functional theory (TDDFT)\nstituents, we designed a series of single-molecule Ir(III) com- calculations (Figure 2b; Figures S1\u2013S6 and Table S1, Support-\nplexes as infrared (IR) two-photon absorption (TPA) PSs with re- ing Information), the weak band extending to 410\u2013467 nm is\nmarkable photothermal conversion e\ufb03ciency (PCE) (Figure 1). attributed to the spin-allowed intraligand (1 IL), or called ligand-\nUsing an 808 nm IR low-power laser (LPL, 100 mW cm\u22122 ), the centered (1 LC), \ud835\udf0b\u2013\ud835\udf0b* transitions on the N\u02c6N ligands, whereas\nmost e\ufb00ective Ir6 was successfully applied in the dual PDT/PTT the two strong bands locate below 350 nm originate from the\nfor malignant melanoma in mice. These \ufb01ndings may contribute ligand-to-ligand charge-transfer (1 LLCT), metal-to-ligand charge-\nto the development of highly e\ufb03cient phototherapeutic drugs for transfer (1 MLCT), 1 IL transitions on the C\u02c6N ligands, and their\nlarge, deeply buried solid tumors in hypoxic environments. respective combinations.\n Residues in the body and cumulative hepatorenal toxicity are The steady state emission spectra of Ir1\u20136 were recorded in\nkey safety concerns for metal-containing drugs.[9] During the in aqueous solution at 25 \u00b0C, and the emission wavelengths (\ud835\udf06em )\nvivo investigation, we attempted to identify the quantities of irid- and quantum yields (\u03a6) were summarized in Table 1. Ir1\u20133\nium persisting in various organs of mice after phototherapy. Af- exhibit consistent emission wavelengths, while those of Ir4\u2013\nter intravenous administration, Ir6 was swiftly removed from 6 are quite di\ufb00erent. From the theoretical results by TDDFT\n\n\nAdv. Healthcare Mater. 2023, 12, 2301227 2301227 (2 of 9) \u00a9 2023 Wiley-VCH GmbH\n\f 21922659, 2023, 28, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202301227 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 2. Photophysical, photochemical, and bioactivity-related properties of Ir1\u20136. a) Absorption (solid) and normalized emission spectra (dashed) in\naqueous solution. b) Real space representation of hole (blue) and electron (green) distributions of Ir6 for the main excited state transitions responsible\nfor excitation (Sn), \ufb01rst singlet emission (F1), and triplet emission (T1). Excitation wavelength (nm), excitation energy (eV), and oscillator strength (f)\nfor each hole-to-electron transition are presented (B3LYP/Lanl2DZ, CPCM). The simpli\ufb01ed Jablonski diagraph describes the energy transfer and main\nnonradiative decay (heat release) pathways. c) TPASCs (\ud835\udf0e) of Ir1\u20136 in the range of 720\u2013880 nm. d) The distributions of Ir1\u20136 between n-octanol and\naqueous phases. e) Representative thermal images of Ir6 in aqueous solution (100 \u00d7 10\u22126 m) irradiated by 400 nm LED (50 mW cm\u22122 ) and 808 nm LPL\n(100 mW cm\u22122 ) for 1, 2, 5, and 10 min.\n\n\nTable 1. Photophysical and electrochemistry data for Ir1\u20136.\n\nComplex \ud835\udf06abs a) [nm] (\ud835\udf16 /104 m\u22121 cm\u22121 ) \ud835\udf06em a) [nm] \u03a6a) \ud835\udf0f a) [ns] Eox b) [V] Ered b) [V]\n\nIr1 290 (4.32), 344 (2.79), 467 (0.33) 593 0.104 8.02 0.52, 0.79 \u22121.24, \u22121.58, \u22122.10\nIr2 287 (6.11), 336 (4.39), 410 (0.88) 588 0.085 34.7 0.70, 0.90 \u22121.26, \u22121.67, \u22122.08\nIr3 289 (4.95), 330 (4.16), 411 (0.85) 593 0.050 52.4 0.68, 0.86 \u22121.30, \u22121.73, \u22122.00\nIr4 287 (5.19), 346 (3.08), 447 (0.36) 593 0.085 4.29 0.52, 0.84 \u22121.03, \u22121.42, \u22122.03\nIr5 285 (2.17), 339 (1.45), 428 (0.24) 489 0.066 1.71 0.70, 0.94 \u22121.24, \u22121.34, \u22121.99\nIr6 286 (3.22), 329 (2.80), 431 (0.33) 527 0.022 13.8 0.71, 0.86 \u22121.14, \u22121.48, \u22122.01\na) b)\n In air-saturated aqueous solution at 25 \u00b0C; In air-saturated anhydrous acetonitrile at 25\u00b0C.\n\n\n\n(Figure 2b; Figures S5 and S6, Supporting Information), the nanoseconds), these emissions are credited to \ufb02uorescence from\n 1\nemissions of Ir4\u20136, from either singlet or triplet states, are pro- IL rather than phosphorescence from 3 IL, although they have\nduced by radiation transitions of the IL excited states on the quite identical con\ufb01gurations. Since no phosphorescence life-\nN\u02c6N ligand. This transition involves the introduced heterocycles, time is detected in the microsecond or second window, we postu-\nwhich have di\ufb00erent electronic e\ufb00ects and may give rise to the late that Ir1\u20136 have a dark (nonradiative) 3 IL state. Phosphores-\nwide variations in \ud835\udf06em . For Ir1\u20133, however, there is a singlet emis- cence is mainly manifested under conditions where vibrational\nsion state with a lower energy level and oscillator strength (f) be- relaxation is limited, such as low temperature, solid state, and so\nlow the heterocycle-involved state (Figures S2\u2013S4, Supporting In- on. Under physiological conditions (>25 \u00b0C and in the mobile\nformation). This state has the nature of 1 LLCT from the C\u02c6N lig- single-molecule state), energy transfer (generating 1 O2 for PDT)\nands to the phenanthroline part of the N\u02c6N ligand and 1 MLCT and nonradiative decay (releasing heat for PTT) will become the\nfrom Ir(III) to the same destination. Therefore, Ir1\u20133 show little main dissipation pathways of the 3 IL state energy of iridium com-\ndi\ufb00erence in emission energy since their emission is not a\ufb00ected plexes. The same \ufb01nding holds true for BODIPY-Ir, where the\nby the heterocycles. di\ufb00erence in energy level between the lowest singlet and triplet\n The luminescence lifetime (\ud835\udf0f) of Ir1\u20136 were further examined excited states (F1 and T1) is as low as Ir1\u20136.[4b] Such a dark triplet\nin aqueous solution at 25 \u00b0C. From the rather short lifetimes (in state has been regarded as advantageous for preventing excessive\n\n\n\nAdv. Healthcare Mater. 2023, 12, 2301227 2301227 (3 of 9) \u00a9 2023 Wiley-VCH GmbH\n\f 21922659, 2023, 28, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202301227 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\nloss of excited state energy and maximizing its usage in PDT and Table 2. Bioactivity-related properties of Ir1\u20136.\nPTT.\n Compound \ud835\udf0e 808 a) [GM] \u03a6\u2206400 b) \u03a6\u2206808 b) \ud835\udf02 808 c) [%] log PO/W d)\n\n Ir1 79 0.62 0.026 17.1 >3.00\n2.2. Electrochemistry Ir2 94 0.78 0.063 17.6 1.16\n Ir3 120 0.82 0.078 17.2 0.04\nAccording to previous studies, the oxidation potential of a PS\n Ir4 87 0.74 0.031 17.0 >3.00\nis important for its ROS generation.[4c,10] Thus, the redox prop-\n Ir5 112 0.82 0.070 17.9 1.83\nerties of Ir1\u20136 were evaluated in acetonitrile (MeCN) by cyclic\nvoltammetry using the Fc+ /Fc couple as the internal standard. Ir6 142 0.96 0.110 18.0 0.06\n a)\nThree irreversible reduction processes were observed for Ir1\u20136, Two-photon absorption cross-section at 808 nm (1 GM = 10\u221250 cm4 s photon\u22121 );\n b)\ncorresponding to the reductions of one N\u02c6N and two C\u02c6N lig- Singlet oxygen quantum yield upon 400 nm LED (50 mW cm\u22122 ) and 808 nm LPL\n c)\n (100 mW cm\u22122 ) irradiation in aqueous solution; Photothermal conversion e\ufb03-\nands, as seen for similar Ir(III) complexes.[11] Two oxidation pro- d)\n ciency at 808 nm; Oil/water partition coe\ufb03cient (standard deviation < 5%).\ncesses were discovered, one irreversible and one reversible. The\nreduction and oxidation potentials (Ered and Eox ) were listed in\nTable 1. Since the HOMOs and LUMOs of Ir1\u20136 were both pop-\nulated on the N\u02c6N ligand, the \ufb01rst oxidation was expected to oc- photon counting technic (detailed in the Supporting Informa-\ncur on the N\u02c6N ligand, while the second oxidation was attributed tion), the 1 O2 quantum yields under 400 and 808 nm excitation\nto Ir(IV)/Ir(III) oxidation owing to the Ir(III) centered HOMO-1 (\u03a6\u2206400 and \u03a6\u2206808 , Table 2) could be calculated by taking into ac-\nand the typical values of Eox for Ir(IV)/Ir(III) in cyclometalated count the di\ufb00erence in photon numbers of excitation lights of\nIr(III) complexes (0.8\u20131.0 V). This provides more evidence that various wavelengths. Ir4\u20136 with fppy C\u02c6N ligands showed higher\nligand-centered HOMO and low-energy IL excited states readily \u03a6\u2206 values than Ir1\u20133 with ppy C\u02c6N ligands and the same N\u02c6N\nparticipate in the electronic/energy transfer of PS. ligand. Ir1 and Ir4 containing bismethylpiperazine (BMP) exhib-\n ited higher \u03a6\u2206 than Ir2 and Ir5 bearing monomethylpiperazine\n (MMP), followed by Ir3 and Ir6 with morpholine (MP).\n2.3. Two-Photon Absorption The \u03a6\u2206400 value is 0.96 for Ir6 in aqueous solution, indicating a\n high e\ufb03ciency with which the energy of the excited state is trans-\nExcitation at 405 nm enabled the aforementioned research on ferred to 1 O2 through 3 IL during OP excitation. In cases of TP ex-\nexcited state characteristics. Due to its low tissue penetration, citation, \u03a6\u2206808 values are apparently decreased. First, this is due\nvisible light with such a short wavelength has limited applica- to the TPA e\ufb03ciency barrier, which means that not all 808 nm\ntions in phototherapy. In addition, in the absence of any PS, this photons can be simultaneously absorbed in pairs and perform\nhigh-energy irradiation can generate biological damage such as the excitation function. Second, the use of a low-energy infrared\nDNA photocleavage, cell death, eye and skin damage, etc. Bene\ufb01t- laser further reduces excitation e\ufb03ciency, albeit increasing the\ning from their exceptional TPA capabilities, Ir(III)-PSs can have feasibility and safety of TP phototherapy. Finally, the upper limit\ntheir excitation wavelength nearly doubled to 800 nm. There- of \u03a6\u2206808 should be 0.5, according to the de\ufb01nition of quantum\nfore, the TPA capacity and excitability of Ir(III)-PSs are crucial yield, since TP excitation requires the absorption of two photons\nfor their application in phototherapy. The TPA cross-sections (\ud835\udf0e) by the PS to produce one 1 O2 . The \u03a6\u2206808 value of Ir1\u20136 are quite\nof Ir1\u20136 have been determined by two-photon (TP) excited \ufb02uo- remarkable, in comparison to [Ru(bpy)3 ]2+ (0.006) and a serious\nrescence. All Ir(III) complexes produce distinct \ufb02uorescence sig- of dinuclear Ru(II) PSs (0.020\u20130.156),[6] under identical experi-\nnals in the range of 720\u2013840 nm upon TP excitation (Figure 2c). mental conditions. The negative correlation between \u03a6\u2206 and \u03a6\nTPA maxima are detected between 740 and 760 nm with remark- is another notable observation. This might imply that radiative\nable \ud835\udf0e values between 357 and 415 GM. The \ud835\udf0e values at 808 nm decay and energy transfer have a compensatory relationship. Ir6,\n(\ud835\udf0e 808 ) are 79\u2013142 GM, which are higher than similar mononu- with the lowest \ufb02uorescence emission intensity and the highest\nclear Ir(III)[11b,12] and Ru(II)[13] complexes at around 800 nm but \u03a6\u2206808 , may become the most promising PS of the series.\nlower than certain dinuclear Ir(III),[14] Ru(II),[6,15] and Os(II)[7]\ncomplexes. Therefore, Ir1\u20136 can be expected to be potent PSs that\ncan be excited by the 808 nm LPL. 2.5. Photothermal Conversion E\ufb03ciency\n\n Given the unique advantages of solid materials and IR in pho-\n2.4. Singlet Oxygen Quantum Yield tothermal conversion (PTC), some Ir(III) complex-containing\n nanoparticles (NPs) and aggregates exhibit excellent PCE at\nThe 1 O2 generation e\ufb03ciency of a PS plays an essential role in 808 nm, despite the fact that their Ir(III) components lack PTC\nits photocytotoxicity. The 1 O2 quantum yields (\u03a6\u0394 ) of Ir1\u20136 in capacity as monomers.[4b,c,16] Appropriate structural modi\ufb01ca-\naqueous solution have been quanti\ufb01ed by a 1 O2 trapping probe, tion and the inclusion of rotatable substituent groups may sig-\n9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA). As ni\ufb01cantly enhance the PCE of non-emitting BODIPY-type pho-\ndepicted in Figures S7 and S8 (Supporting Information), the ab- tothermal agent (PTA) and BODIPY-encapsulated NP to above\nsorbance of Ir(III) complex-containing ABDA decreased rapidly 88%.[16b,c,17] Undoubtedly, \ufb01nding out if this strategy may be em-\nunder irradiation with 400 nm LED (50 mW cm\u22122 ) and 808 nm ployed as well for constructing single-molecule Ir(III)-based dual\nLPL (100 mW cm\u22122 ), indicating the generation of 1 O2 . Using a TPA-PS/PTA is a worthy endeavor.\n\n\nAdv. Healthcare Mater. 2023, 12, 2301227 2301227 (4 of 9) \u00a9 2023 Wiley-VCH GmbH\n\f 21922659, 2023, 28, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202301227 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 3. Cellular localization, uptake, photoinduced apoptosis, and mechanism of Ir(III) complexes. a) Subcellular colocalization of Ir6 with MitoTracker\nGreen (MTG) and Hoechst 33324 (Hoechst) in A375 human melanoma cells by confocal laser scanning microscopy (CLSM) upon one-photon (OP,\n405 nm) and two-photon (TP, 808 nm) excitations (scale bar: 20 \u03bcm). b) The distribution of iridium for Ir1\u20136 in various regions of A375 cells as determined\nby inductively coupled plasma mass spectrometry (ICP-MS). Data represent the mean \u00b1 SD (n = 3). c) Relative uptake amount of Ir4\u20136 in the presence\nof endocytic inhibitor chlorpromazine (CPZ, 10.0 \u03bcg mL\u22121 ), amiloride (100.0 \u03bcg mL\u22121 ), or nystatin (50.0 \u03bcg mL\u22121 ) at 37 \u00b0C or without inhibitor at 4 \u00b0C.\nData represent the mean \u00b1 SD (n = 3). d) DCF-indicated intracellular 1 O2 generation by Ir6 upon TP excitations (scale bar: 50 \u03bcm) and its suppression\nby 1 O2 scavenger ABDA (100 \u00d7 10\u22126 m). e) Flow cytometry detection (stained with Annexin V-FITC and PI) of photoinduced apoptosis in A375 cells\ninduced by Ir(III) complexes in the absence and presence of 808 nm LPL (100 mW cm\u22122 ) irradiation. f) Caspase-3 and -9 activity in A375 cells after\ntreatment with PBS (control) and Ir(III) complexes in the dark and under 808 nm LPL (100 mW cm\u22122 ) irradiation. Data represent the mean \u00b1 SD (n =\n3).\n\n\n The PCEs of Ir1\u20136 were measured in aqueous solution. When substituent-dependent lipophilicity (Table 2). Ir1 and Ir4 with MP\nexposed to 808 nm LPL, the temperatures of Ir1\u20136 solution (100 \u00d7 group exhibited signi\ufb01cant log PO/W values greater than 3.0 and\n10\u22126 m) steadily increased with \u0394T = 13.3\u201314.2 \u00b0C (Figure 2e and extremely poor solubilities in aqueous solution (20 \u00d7 10\u22126 m, 5%\nFigure S9, Supporting Information). The amplitude of the tem- DMSO), which may be disadvantaged to their drug utilization.\nperature increase has a strong concentration dependence. After Ir2 and Ir5 with MMP group showed moderate lipophilicity and\nfour cycles of heating/cooling, the performance has remained better solubility (50 \u00d7 10\u22126 m, 5% DMSO). Ir3 and Ir6 with BMP\nlargely unaltered. The PCEs of Ir1\u20136 at 808 nm (\ud835\udf02 808 , Table 2) group had an optimum hydrophilic and hydrophobic balance and\nshow no discernible individual di\ufb00erences (17.0%\u201318.0%), in- quite high solubility (>100 \u00d7 10\u22126 m without DMSO), which is\ndicating that the complexes release heat primarily through the bene\ufb01cial in the process of pharmaceutical application, such as\nrich and diverse vibration modes of the \ufb02exible and mobile six- a balanced volume of distribution and the potential for good ab-\nmembered ring, while the rotation of the methyl substituents sorption and bioavailability.[18]\nmay not play a signi\ufb01cant role. In contrast, analogues of Ir1\u2013\n6 containing rigid conjugated ligands have no PTC activity un-\nder the same conditions,[11b] highlighting the crucial role of the 2.7. Subcellular Localization and Cell Uptake\nsix-membered ring in heat release. The PCEs of Ir1\u20136 are lower\nthan those of the self-assembled aggregates of IrDAD (27.5%, The e\ufb00ectiveness of PDT and PTT depends heavily on the cell\n808 nm laser, 700 mW cm\u22122 ), in which aggregation played an uptake and intracellular localization of PSs and PTAs. As shown\nessential part in the PTC.[4c] Despite having a 50% greater PCE in Figure 3a and Figure S15 (Supporting Information), all Ir(III)\nthan Ir1\u20136, IrDAD has lost the ability to generate 1 O2 in a single- complexes showed red luminescence within cells. For hydropho-\nmolecule state. Ir1\u20136 are therefore the only example of single- bic Ir1, Ir2, Ir4, and Ir5, the complex precipitated around or in-\nmolecule Ir(III) complexes with both the capacity to produce 1 O2 side the cell at concentrations that did not reach their saturated\nand the PTC activity at 808 nm, supporting their application as solubility. This suggests that some parts of the cell can enrich\ndual PDT/PTT reagents. and locally supersaturate the complex. For amphiphilic Ir3 and\n Ir6, no precipitation was generated. The signals of Ir3 and Ir6\n matched well with the mitochondrial dye MTG, suggesting that\n2.6. Lipophilicity and Solubility they target mitochondria in cells.\n The cell uptake and pathway of Ir1\u20136 were further investi-\nFigure 2d and Figure S10 (Supporting Information) show the gated. ICP-MS \ufb01ndings suggest that at the same concentration\nlipophilicity (log PO/W values) of Ir1\u20136. The complexes displayed of complex, Ir6 was taken up by cells the most (Figure 3b). Its\n\n\nAdv. Healthcare Mater. 2023, 12, 2301227 2301227 (5 of 9) \u00a9 2023 Wiley-VCH GmbH\n\f 21922659, 2023, 28, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202301227 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\nTable 3. In vitro cytotoxicities (IC50 , \u03bcm) of Ir(III) complexes and cisplatin against A375 cell lines under various conditions.\n\nCompound Darka) TPb) PIc) TP+Ascd) TP+Icee) TP+Asc+Icef)\n\nIr1 >20 14.2 \u00b1 1.1 >1.41 >20 >20 >20\nIr2 >50 4.12 \u00b1 0.27 >12.1 44.2 \u00b1 5.6 38.4 \u00b1 4.9 >50\nIr3 134 \u00b1 11 3.10 \u00b1 0.21 43.3 76.0 \u00b1 3.1 22.5 \u00b1 2.8 115 \u00b1 10\nIr4 >20 17.8 \u00b1 1.3 >1.12 >20 >20 >20\nIr5 >50 11.3 \u00b1 1.0 >4.42 >50 >50 >50\nIr6 113 \u00b1 10 0.347 \u00b1 0.022 325 58.5 \u00b1 4.9 13.6 \u00b1 0.50 98.6 \u00b1 8.1\nCisplatin 1.62 \u00b1 0.12 1.36 \u00b1 0.11 1.19 1.39 \u00b1 0.11 1.42 \u00b1 0.12 1.53 \u00b1 0.13\na) b) c)\n Under dark condition; Upon two-photon (TP) excitation by an 808 nm LPL (100 mW cm\u22122 , light dose = 30.0 J cm\u22122 ); Phototherapy indexes (PIs) under TP excitation;\nd) \u22123 e) f) \u22123\n TP excitation in the presence of ascorbic acid (Asc, 1.0 \u00d7 10 m); TP excitation in an ice bath; TP excitation in an ice bath with Asc (1.0 \u00d7 10 m). Data represent the\nmean \u00b1 SD (n = 6).\n\n\n\ndistribution in mitochondria accounted for up to 95% of the total decreased photocytotoxicity for Ir(III) complexes. Simultaneous\ncellular uptake. Ir6 showed decreased uptake in the presence of inhibition of PDT and PTT e\ufb00ects led to an almost complete loss\nthe clathrin inhibitor chlorpromazine (CPZ) and at 4\u00b0C, indicat- of photocytotoxicity. Hence, their strong photocytotoxicity results\ning clathrin-mediated and energy-dependent pathways. The up- from a combined PDT/PTT e\ufb00ect. In addition, the toxicity of PDT\ntake of Ir4 and Ir5 was inhibited by nystatin and at 4 \u00b0C, indicat- or PTT alone was signi\ufb01cantly lower than that of dual PDT/PTT,\ning that they entered the cell via caveolae-mediated and energy- indicating a synergistic e\ufb00ect between the two mechanisms. Ir6\ndependent pathways. It is interesting that slight modi\ufb01cations in thus functions as a powerful dual PS/PTA with minimal cytotox-\nligand structures lead to di\ufb00erent endocytic pathways of Ir(III) icity in the dark.\ncomplexes. Ir6, which can penetrate cells e\ufb00ectively, may per-\nform better in antitumor phototherapy.\n 2.10. Apoptosis Assay and Caspase Activation\n\n2.8. Intracellular 1 O2 Generation Photogenerated 1 O2 triggers the mitochondrial pathway of apop-\n tosis in melanoma cells.[19] Apoptosis of A375 cell lines induced\nIntracellular 1 O2 generation of Ir6 was analyzed using the ROS by Ir1\u2013Ir6 during dual PDT/PTT were investigated by Annexin\ndetector DCFH-DA (2\u2032,7\u2032-dichlorodihydro\ufb02uorescein diacetate, V-FITC/propidium iodide (PI) staining (Figure S13, Support-\nFigure 3d). No ROS was identi\ufb01ed in the absence of Ir6 (PBS+TP) ing Information). The data for each quadrant were illustrated\nor in the dark (Ir6+Dark). Strong DCF (dichloro\ufb02uorescein) sig- in Figure 3e. Under dark conditions, A375 cells treated with\nnals have been observed in the presence of Ir6 upon TP excitation Ir(III) complex showed patterns quite comparable to those of\n(Ir6+TP), showing e\ufb03cient production of ROS. The almost com- blank cells. Upon irradiation, the late apoptotic fractions of Ir(III)\nplete suppression of DCH \ufb02uorescence upon addition of the 1 O2 complex-treated cells increased, particularly for Ir6, suggesting\nscavenger ABDA (Ir6+TP+ABDA) indicates that 1 O2 is the major that they may promote apoptosis in tumor cells. In the apoptotic\nROS generated by Ir6. process, caspase-9 propagates a cascade of further caspase pro-\n cessing events by directly cleaving and activating caspase-3 and\n caspase-7 to execute cell death.[20] Caspase-3 and -9 activity was\n2.9. In Vitro Cytotoxicity measured in A375 cells treated with Ir(III) complexes (Figure 3f).\n All complexes exhibited no caspase-3 or -9 activation under\nUnder dark conditions, Ir3 and Ir6 displayed fairly low toxicity dark conditions. With 808 nm LPL irradiation, both caspase-\n(IC50 > 100 \u00d7 10\u22126 m) to A375 cells, as determined by the CCK-8 3 and -9 activity rose substantially. These \ufb01ndings demon-\nassay (Table 3). Ir1, Ir2, Ir4, and Ir5 did not inhibit cell viability at strate that Ir(III) complexes initiate a photo-dependent apoptosis\ntheir respective limits of solubility (20 or 50 \u03bcM\u00d7 10\u22126 m). Since mechanism.\n400 nm irradiation directly induces cell damage, only 808 nm Although laser-irradiated \u201chot\u201d NPs stimulate the intrin-\nLPL irradiation was used to assess photocytotoxicity. Ir1\u2212Ir6 ex- sic/mitochondrial pathway of apoptosis,[21] no single-molecule\nhibited notable photocytotoxicities. Ir6 achieved a phototherapy has been identi\ufb01ed to trigger apoptosis by PTC. The caspase ac-\nindex (PI) of 325, ranking it among the most e\ufb00ective photothera- tivity of Ir6-treated A375 cells decreased greatly in the presence of\npeutic molecules and materials recorded to date. As a chemother- Asc (Figure 3f), due to the clearance of 1 O2 . Nonetheless, the ac-\napeutic drug, cisplatin showed no signi\ufb01cant change in toxicity tivities remained higher than 1, suggesting that the apoptosis was\n(PI = 1.15\u20131.62) under either dark or irradiation conditions. To not entirely dependent on 1 O2 . In addition, when the cells were ir-\nreveal the relative contributions of PDT and PTT to the photocyto- radiated in an ice bath, caspase activity also decreased. Therefore,\ntoxicities of Ir1\u2013Ir6, cytotoxicities were examined in the presence besides 1 O2 , the heat released by irradiated Ir6 also contributed to\nof a ROS scavenger, ascorbic acid (Asc, 1.0 \u00d7 10\u22123 m), or in an the activation of caspase and cell apoptosis. This favorable char-\nice bath.[4b] Due to the elimination of PDT and PTT activity, the acteristic o\ufb00ers crucial support for the development of Ir6 as a\naddition of Asc and treatment with an ice bath during irradiation highly e\ufb00ective dual PDT/PTT reagent.\n\n\nAdv. Healthcare Mater. 2023, 12, 2301227 2301227 (6 of 9) \u00a9 2023 Wiley-VCH GmbH\n\f 21922659, 2023, 28, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202301227 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n\n\n\nFigure 4. In vivo dual PDT/PTT assay and hepato-/nephrotoxicity evaluation of Ir6. a) Real-time thermal images of BALB/c nude mice (subcutaneous\nA375 human melanoma xenograft model) before and after the in vivo dual PDT/PTT assay (n = 5). b) Tumor growth curves of the four groups after\nvarious treatments (n = 5). c) Body weight curves of the four groups after various treatments (n = 5). d) Photograph of representative post-treatment\nsolid tumors for each group (n = 5). e) Iridium amounts in the collected organs in Ir6-injected groups (Group III and IV, 14 d after injection, n = 5).\nND: not detected. The amount of iridium administered was calculated by the total body weight. Cell viability of f) human malignant melanoma cell line\nA375, g) human normal liver cell line HL-7702, and h) human normal kidney cell line HK-2 incubated with various dose of Ir6 and cisplatin (CP) for 24 h\n([drug] = 0\u201210 \u00d7 10\u22126 m). Data represent the mean \u00b1 SD (n = 6).\n\n\n2.11. In Vivo Dual PDT/PTT The di\ufb00erent hematoxylin and eosin (H&E) staining pattern of\n Group IV tumor sections compared to the others suggests that\nA375 tumor-bearing mice (BALB/c mouse model with tumor Ir6-mediated dual PDT/PTT can cause damage to tumor tissues,\nxenografts) of di\ufb00erent drug groups were intratumorally injected whereas no appreciable lesions were observed in the H&E stain-\nwith Ir6, irradiated with 808 nm LPL (Group IV, 0.1 W cm\u22122 , light ing of sections from the major organs (Figure S14, Supporting\ndose = 30.0 J cm\u22122 ) or untreated (Group III). The control groups Information), con\ufb01rming the nontoxic nature of Ir6 in vivo.\nreceived PBS injections and were handled both with and with-\nout irradiation (Group II and I). The body temperatures of mice\nwere tracked by IR thermal images. After irradiation, the tem- 2.12. Biodistribution and Hepato-/Nephrotoxicity Evaluation\nperature of tumor sites in mice (Group IV) rose to 47.9 \u00b0C (\u2206T\n= 13.7 \u00b0C, Figure 4a), while \u2206T values in the other groups were The toxicity of heavy metals to the human body, particularly hepa-\n<1.0 \u00b0C. A temperature exceeding 41 \u00b0C is considered e\ufb00ective totoxicity and nephrotoxicity, is a major safety concern for metal-\nfor destroying tumor tissue.[28] No skin injury occurred during containing drugs. While studies have focused on the e\ufb00ective-\nphototherapy, since the irradiation of 808 nm LPL in this study ness of drug distribution to tumors over hours after injection into\nwas below 30% of the MPE standard for laser exposure to skin animals, residues and potential toxicity after treatment have been\n(0.33 W cm\u22122 ). After 14 d, the tumor mean volume (V) of Groups largely overlooked. Here, we used ICP-MS to analyze the amount\nI\u2013III reached 2000 mm3 (Figure 4b), and there was no loss of of Ir residues in the collected organs and tumors (Figure 4e) from\nbody weight in the mice (Figure 4c), indicating that the systemic the Ir6-injected mice at the end of the in vivo assay (14 d after\ntoxicity of Ir6 in mice without irradiation is extremely low. The ir- administration). Unsurprisingly, trace quantities of Ir were de-\nradiation group (Group IV) showed a substantial antitumor e\ufb00ect tected in tumors, kidneys, and livers, but none in other organs.\nwith a value of 310 mm3 and an inhibition rate of 85% (Figure 4d). Ir6 can be cleared from the body more e\ufb03ciently than cisplatin\n\n\nAdv. Healthcare Mater. 2023, 12, 2301227 2301227 (7 of 9) \u00a9 2023 Wiley-VCH GmbH\n\f 21922659, 2023, 28, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202301227 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.advhealthmat.de\n\n(t1/2 = 58\u201373 h), Por\ufb01mer Sodium (t1/2 = 250 h), and Photofrin II Acknowledgements\n(t1/2 > 100 h), as shown by the mean total residue of 0.85% in ten\nIr6-injected mice (estimated t1/2 = 48 h). A short half-life is also S.-J.T. and Q.-F.L. contributed equally to this work. This work was sup-\n ported by the National Natural Science Foundation of China (22167022),\nbene\ufb01cial for the rapid excretion of the PS following photother- Yunnan Provincial Science and Technology Department (2018FB022), and\napy to reduce side e\ufb00ects such as photodermatosis. Youth Talents Project of Yunnan Province (2018-057). The authors thank\n In order to verify whether the residue is toxic to the liver and the Advanced Analysis and Measurement Center of Yunnan University for\nkidney, the cytotoxicities of Ir6 and cisplatin to human normal their help in characterization.\nliver (HL-7702) and kidney (HK-2) cells have been further stud-\nied. In vitro cytotoxicity studies have demonstrated that Ir6 has\nminimal cytotoxicity to A375 cells in the absence of irradiation, Con\ufb02ict of Interest\nwhereas cisplatin can signi\ufb01cantly inhibit the growth of A375\n The authors declare no con\ufb02ict of interest.\ncells (Figure 4f). In the case of HL-7702 and HK-2 cells, cisplatin\nalso exhibited similar toxicity. In contrast, Ir6 was much less toxic\nto these two normal cells. At concentrations of 1\u201310 \u00d7 10\u22126 m,\nwhich is 5\u20136 orders of magnitude higher than the total residue Data Availability Statement\nof Ir6 in mice, normal cell viability remained greater than 99% The data that support the \ufb01ndings of this study are available from the cor-\nto 93% (Figure 4g,h). This allows Ir6 to reach a level of relative responding author upon reasonable request.\nsafety prior to its total removal from the body.\n\n Keywords\n3. Conclusions antitumor therapy, iridium complexes, photodynamic therapy, photother-\n mal therapy\nIn conclusion, by rational design and distribution of excited state\nenergy, a series of single-molecule Ir(III) complexes have been Received: April 26, 2023\nendowed with the capability to act as infrared two-photon absorp- Revised: May 30, 2023\ntion, dual PDT and PTT reagents using an 808 nm low-power Published online: June 20, 2023\nlaser. Ir6, the most potent complex in the series, has exhibited a\nremarkable phototherapy index in vitro and dual PDT and PTT\nactivity for malignant melanoma in vivo. Ir6 also has minimal\nhepato-/nephrotoxicity in vitro and can be eliminated from the [1] a) L. Zhang, N. Montesdeoca, J. Karges, H. Xiao, Angew. Chem., Int.\nbody e\ufb00ectively. These \ufb01ndings may contribute to the develop- Ed. 2023, 62, e202300662; b) S. Sen, M. Won, M. S. Levine, Y. Noh,\n A. C. Sedgwick, J. S. Kim, J. L. Sessler, J. F. Arambula, Chem. Soc.\nment of highly e\ufb03cient phototherapeutic drugs for large, deeply\n Rev. 2022, 51, 1212; c) J. Karges, Angew. Chem., Int. Ed. 2022, 61,\nburied solid tumors in hypoxic environments.\n e202112236.\n [2] a) Z. S. Yang, Y. H. Yao, A. C. Sedgwick, C. C. Li, Y. Xia, Y. Wang, L.\n Kang, H. M. Su, B. W. Wang, S. Gao, J. L. Sessler, J. L. Zhang, Chem.\n4. Experimental Section Sci. 2020, 11, 8204; b) J. Du, T. Shi, S. Long, P. Chen, W. Sun, J. Fan,\n X. Peng, Coord. 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