Dual-Emissive Iridium(III) Complexes and Their Applications in Biological Sensing and Imaging.

Phosphorescent iridium(III) complexes are widely recognized for their unique properties in the excited triplet state, making them crucial for various applications including biological sensing and imaging. Most of these complexes display single phosphorescence emission from the lowest-lying triplet state after undergoing highly efficient intersystem crossing (ISC) and ultrafast internal conversion (IC) processes. However, in cases where these excited-state processes are restricted, the less common phenomenon of dual emission has been observed. This dual emission phenomenon presents an opportunity for developing biological probes and imaging agents with multiple emission bands of different wavelengths. Compared to intensity-based biosensing, where the existence and concentration of an analyte are indicated by the brightness of the probe, the emission profile response involves modifications in emission color. This enables quantification by utilizing the intensity ratio of different wavelengths, which is self-calibrating and unaffected by the probe concentration and excitation laser power. Moreover, dual-emissive probes have the potential to demonstrate distinct responses to multiple analytes at separate wavelengths, providing orthogonal detection capabilities. In this concept, we focus on iridium(III) complexes displaying fluorescence-phosphorescence or phosphorescence-phosphorescence dual emission, along with their applications as biological probes for sensing and imaging.

Cyclometalated Iridium(III) Complexes Containing Viologen-Modified Phenylpyridine Ligands as Electroluminochromic Active Molecules for Information Display.

Electroluminochromic (ELC) materials have garnered significant research interest because of their potential applications in lighting, displaying, and sensing. These materials exhibit reversible modulation of photoluminescence under low-voltage stimuli. Here five phosphorescent iridium(III) complexes are reported featuring viologen-substituted 2-phenylpyridine (Vppy) ligands acting as electroactive components. Four of the complexes are bis-cyclometalated and coordinated with either neutral bipyridine derivatives or negatively charged 2-picolinate. The remaining complex is heteroleptic tris-cyclometalated, containing one Vppy and two 2-phenylquinoline ligands. Upon photoexcitation, the bis-cyclometalated complexes exhibit orange to red phosphorescence originating from mixed triplet metal-to-ligand charge transfer (3MLCT) and intraligand (3IL) dπ(Ir)/π(Vppy) → π*(Vppy) state, whereas the tris-cyclometalated complex is non-emissive due to a low Ir(IV/III) oxidation potential favoring oxidative quenching by the viologen pendants. When the cationic viologens are electrochemically reduced to their neutral form, the bis-cyclometalated complexes show a remarkable blue-shift in their phosphorescence maxima due to increased energy levels of the Vppy molecular orbitals. In the case of the tris-cyclometalated complex, reduction of the viologen groups interrupts the quenching process, leading to a luminescence turn-on. These complexes are used to develop ELC devices, which exhibit reversible luminescence response in terms of color or on-off switching under a low voltage of 2 V.

Bioorthogonal dissociative rhenium(i) photosensitisers for controlled immunogenic cell death induction.

Photosensitisers for photoimmunotherapy with high spatiotemporal controllability are rare. In this work, we designed rhenium(i) polypyridine complexes modified with a tetrazine unit via a bioorthogonally activatable carbamate linker as bioorthogonally dissociative photosensitisers for the controlled induction of immunogenic cell death (ICD). The complexes displayed increased emission intensities and singlet oxygen (1O2) generation efficiencies upon reaction with trans-cyclooct-4-enol (TCO-OH) due to the separation of the quenching tetrazine unit from the rhenium(i) polypyridine core. One of the complexes containing a poly(ethylene glycol) (PEG) group exhibited negligible dark cytotoxicity but showed greatly enhanced (photo)cytotoxic activity towards TCO-OH-pretreated cells upon light irradiation. The reason is that TCO-OH allowed the synergistic release of the more cytotoxic rhenium(i) aminomethylpyridine complex and increased 1O2 generation. Importantly, the treatment induced a cascade of events, including lysosomal dysfunction, autophagy suppression and ICD. To the best of our knowledge, this is the very first example of using bioorthogonal dissociation reactions as a trigger to realise photoinduced ICD, opening up new avenues for the development of innovative photoimmunotherapeutic agents.

Luminescent iridium(III) porphyrin complexes as near-infrared-emissive biological probes.

We report herein the design, synthesis and characterisation of a series of luminescent iridium(III) porphyrin complexes [Ir(ttp)(CH2CH2OH)] (H2ttp = 5,10,15,20-tetra-4-tolylporphyrin) (1), [Ir(tpp-Ph-NO2)(CO)Cl] (H2tpp-Ph-NO2 = 5-(4-((4-nitrophenoxy)carbonyloxymethyl)phenyl)-10,15,20-triphenylporphyrin) (2), [Ir(tpp-COOMe)(Py)2](Cl) (H2tpp-COOMe = 5-(4-methoxycarbonylphenyl)-10,15,20-triphenylporphyrin; Py = pyridine) (3) and [Ir(tpp-COOH)(Py)2](Cl) (H2tpp-COOH = 5-(4-carboxylphenyl)-10,15,20-triphenylporphyrin) (4). All the complexes displayed long-lived near-infrared (NIR) emission attributed to an excited state of mixed triplet intraligand (3IL) (π → π*) (porphyrin) and triplet metal-to-ligand charge transfer (3MLCT) (dπ(Ir) → π*(porphyrin)) character. The cytotoxicity of the complexes toward HeLa cells was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cationic complexes 3 and 4 exhibited higher cytotoxic activity toward HeLa cells than their neutral counterparts 1 and 2. Cellular uptake studies by inductively coupled plasma-mass spectrometry (ICP-MS) and laser-scanning confocal microscopy (LSCM) indicated that complexes 3 and 4 showed higher cellular uptake efficiencies than complexes 1 and 2 due to their cationic charge, and they were enriched in the perinuclear region of the cells with negligible nuclear uptake. Additionally, the carboxyl complex 4 was used to label a model protein bovine serum albumin (BSA) via an amidation reaction. The resultant luminescent protein conjugate 4-BSA displayed similar photophysical properties and intracellular localisation behaviour to its parent complex. The results of this work will contribute to the development of luminescent iridium(III) porphyrin complexes and related bioconjugates as NIR-emissive probes for bioimaging applications.

Dual-emissive Iridium(III) Complexes as Phosphorescent Probes with Orthogonal Responses to Analyte Binding and Oxygen Quenching.

Phosphorescent probes often show sensitive response toward analytes at a specific wavelength. However, oxygen quenching usually occurs at the same wavelength and thus hinders the accurate detection of analytes. In this study, we have developed dual-emissive iridium(III) complexes that exhibit phosphorescence responses to copper(II) ions at a wavelength distinct from that where oxygen quenching occurs. The complexes displayed colorimetric phosphorescence response in aqueous solutions under different copper(II) and oxygen conditions. In cellular imaging, variation in oxygen concentration over a large range from 5 % to 80 % can modulate the intensity and lifetime of green phosphorescence without affecting the response of red phosphorescence toward intracellular copper(II) ions.

Cellular imaging properties of phosphorescent iridium(III) complexes substituted with ester or amide groups.

Phosphorescent iridium(III) complexes have been extensively investigated as cellular imaging reagents and sensors. The intracellular localization of the complexes is known to be closely related to their formal charge, molecular size, lipophilicity, and bioactive pendants. Herein, we reported four phosphorescent iridium(III) complexes with the diimine ligands being modified with ester or amide groups as imaging reagents for living cells. The complexes have the same positive charge and very similar molecular size and weight. The lipophilicity of the complexes is similar ranging from 1.45 to 2.14. Upon internalization into living HeLa cells, while complexes 2-4, like most other iridium(III) complexes, were localized in the cytoplasm, complex 1 unexpectedly stained the whole cells including nuclei. The nuclear uptake of complex 1 was not observed when the cells were pretreated with chlorpromazine or nocodazole, suggesting that clathrin and microtubules mediated the nuclear uptake of complex 1. Additionally, the nuclear uptake efficiency is related to the cell division cycle. The complex was mainly concentrated in the nucleus when the cells were in mitosis, and distributed in whole cells when the cells were in the interphases. Furthermore, complex 1 exhibited a longer luminescence lifetime in the nucleus than in the cytoplasm as revealed by photoluminescence lifetime imaging microscopy (PLIM). Incubation of the cells in the hypoxia environment elongated the lifetime of the cytoplasmic complex, but hardly affected the luminescence properties of the intranuclear complex.

Dual-lifetime luminescent probe for time-resolved ratiometric oxygen sensing and imaging.

Fluorescent/phosphorescent dual-emissive polymers or hybrids consisting of both fluorophore and phosphor have been used as self-calibrating probes and imaging reagents for cellular molecular oxygen. Oxygen selectively quenches the phosphorescence and the fluorescence serves as an internal reference. The phosphorescence/fluorescence ratio is used as a quantitative indicator of oxygen content. In wavelength-ratiometric probes, the fluorophore and phosphor are designed to emit at different wavelengths. It is easy to achieve spectral separation, but the phosphorescence/fluorescence ratio fluctuates due to the difference in the absorption and scattering of light at different wavelengths by biological samples. Herein we reported a lifetime-ratiometric luminescent polymeric probe where the fluorophore and phosphor emitted at the same wavelength. Spectral separation was achieved based on the difference in their excited-state lifetimes via time-resolved luminescence analysis and imaging. The probe exhibited a phosphorescence lifetime of about 931 ns with a phosphorescence/fluorescence ratio of 4.49 in deaerated aqueous buffer. The lifetime was shortened to 251 ns and the ratio decreased to 1.08 in oxygen saturated solution because of phosphorescence quenching. The utilization of the probe for quantitative oxygen sensing and mapping in living HeLa cells was demonstrated using calibration curves obtained from fixed cells.

Manipulating Electroluminochromism Behavior of Viologen-Substituted Iridium(III) Complexes through Ligand Engineering for Information Display and Encryption.

Electrically controlling photoluminescence has attracted great research interest and offers many opportunities for technological developments. Electroluminochromic materials undergo redox reactions under low-voltage stimuli to achieve reversible luminescence switching. Till now, photoluminescence switching of a single molecule caused by electrical stimuli is restricted to intensity response because the redox-active moieties are good electron donors or acceptors and electrical stimuli can regulate the photoinduced electron-transfer and affect the luminescence intensity. In this work, the manipulation of the electroluminochromism behavior of a series of viologen-substituted iridium(III) complexes through the regulation of ligand orbital energy levels and electronic communication between the viologen pendants and the iridium(III) complex core is reported. Electrochemical redox reactions reversibly modulate either the luminescence quenching effect or the push-pull electronic effect of the viologen substituents, achieving multicolor "on-off" luminescence response toward electrical stimuli and luminescence manipulation between two emissive states with different wavelengths and lifetimes. To illustrate the promising applications of these electroluminochromic materials, recording and displaying luminescence information under electrical stimuli are demonstrated. Information encryption is realized by letting the electroluminochromism occur in the near-infrared region or in the time domain. Near-infrared camera or time-resolved luminescence analysis can be used to help read the invisible information.

Time-resolved analysis of photoluminescence at a single wavelength for ratiometric and multiplex biosensing and bioimaging.

Simultaneous analysis of luminescence signals of multiple probes can improve the accuracy and efficiency of biosensing and bioimaging. Analysis of multiple signals at different wavelengths usually suffers from spectral overlap, possible energy transfer, and difference in detection efficiency. Herein, we reported a polymeric luminescent probe, which was composed of a phenothiazine-based fluorescent compound and a phosphorescent iridium(iii) complex. Both luminophores emitted at around 600 nm but their luminescence lifetimes are 160 times different, allowing time-resolved independent analysis. As the fluorescence was enhanced in response to oxidation by hypochlorite and the phosphorescence was sensitive toward oxygen quenching, a four-dimensional relationship between luminescence intensity, fluorescence/phosphorescence ratio, hypochlorite concentration, and oxygen content was established. In cellular imaging, time-resolved photoluminescence imaging microscopy clearly showed the independent fluorescence response toward hypochlorite and phosphorescence response toward oxygen in separated time intervals. This work opens up a new idea for the development of multiplex biosensing and bioimaging.

Cyclometalated iridium(III) complexes containing an anthracene unit for sensing and imaging singlet oxygen in cellular mitochondria.

Singlet oxygen (1O2), as a highly reactive oxygen species, plays an important role in the physical, chemical and biomedical fields, especially during photodynamic therapy (PDT) process. In this work, two iridium(III) complexes containing an anthracene unit in their diimine ligand were designed and synthesized to monitor 1O2 in living cells. The complexes were weakly emissive owing to the photoinduced electron transfer process, but exhibited intense luminescence upon capturing 1O2, resulting from the formation of the corresponding endoperoxide analogues. The remarkable turn-on luminescence response was specific toward 1O2 and in preference to other reactive oxygen species. The utilization of one of the complexes for imaging 1O2 in living cells has also been demonstrated using three different cells lines. Cells incubated with the complexes were hardly emissive. Further light irradiation at 475 nm triggered intracellular emission turn on, indicative of the production of 1O2 photochemically. The emissive pattern was well colocalized with commercially available MitoTracker, suggesting the potential applications of the complexes for imaging mitochondria 1O2. The 1O2 capturing properties rendered the complexes low photocytotoxicity since 1O2-caused oxidative damage toward cellular molecules and structures was inhibited.

Thermally activated triplet exciton release for highly efficient tri-mode organic afterglow.

Developing high-efficient afterglow from metal-free organic molecules remains a formidable challenge due to the intrinsically spin-forbidden phosphorescence emission nature of organic afterglow, and only a few examples exhibit afterglow efficiency over 10%. Here, we demonstrate that the organic afterglow can be enhanced dramatically by thermally activated processes to release the excitons on the stabilized triplet state (T1*) to the lowest triplet state (T1) and to the singlet excited state (S1) for spin-allowed emission. Designed in a twisted donor-acceptor architecture with small singlet-triplet splitting energy and shallow exciton trapping depth, the thermally activated organic afterglow shows an efficiency up to 45%. This afterglow is an extraordinary tri-mode emission at room temperature from the radiative decays of S1, T1, and T1*. With the highest afterglow efficiency reported so far, the tri-mode afterglow represents an important concept advance in designing high-efficient organic afterglow materials through facilitating thermally activated release of stabilized triplet excitons.

Bioorthogonal "Labeling after Recognition" Affording an FRET-Based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

Bis-labeling with a luminescent energy donor/acceptor pair onto biological substrates affords probes which give FRET readouts for the detection of interaction partners. However, the covalently bound luminophores bring about steric hindrance and nonspecific interaction, which probably perturb the biological recognition. Herein, we designed a highly sensitive and specific "labeling after recognition" sensing approach, where luminophore labeling occurred after the biological recognition. Taking the cutting enzyme caspase-3 as an example, we demonstrated the detection of its catalytic activity in solution and apoptotic cells using the tetrapeptide motif Asp-Glu-Val-Asp (DEVD) as the cleavable substrate, and an iridium(III) complex and a rhodamine derivative as the energy donor/acceptor pair. The DEVD tetrapeptide was modified with an azide and a GK-norbornylene groups at the amino and carboxyl terminuses, respectively, which allowed donor/acceptor bis-labeling via two independent catalysis-free bioorthogonal reactions. The phosphorescence lifetime of the iridium(III) complex was quenched upon bis-labeling owing to the intracellular FRET to the rhodamine derivative, and significantly elongated upon the peptide being catalytically cleaved by caspase-3. Interestingly, the sensitivity and efficiency of the lifetime responses were much higher in the "labeling after recognition" sensing approach. Molecular docking analysis showed that the steric hindrance and nonspecific interactions partially inhibited the biological recognition of the DEVD substrate by caspase-3. The imaging of the catalytic activity of caspase-3 in apoptotic cells was demonstrated via photoluminescence lifetime imaging microscopy. Lifetime analysis not only confirmed the occurrence of intracellular bioorthogonal bis-labeling and catalytic cleavage, but also showed the extent to which the two dynamic processes occurred.

De Novo Design of Polymeric Carrier to Photothermally Release Singlet Oxygen for Hypoxic Tumor Treatment.

Intratumoral hypoxia extremely limits the clinic applications of photodynamic therapy (PDT). Endoperoxides allow thermally releasing singlet oxygen (1O2) in a defined quantity and offer promising opportunities for oxygen-independent PDT treatment of hypoxic tumors. However, previous composite systems by combining endoperoxides with photothermal reagents may result in unpredicted side effects and potential harmful impacts during therapy in vivo. Herein, we de novo design an all-in-one polymer carrier, which can photothermally release 1O2. The strategy has been demonstrated to effectively enhance the production of 1O2 and realize the photodamage in vitro, especially in hypoxic environment. Additionally, the polymer carrier accumulates into tumor after intravenous injection via the enhanced permeation and retention effects and accelerates the oxygen-independent generation of 1O2 in tumors. The oxidative damage results in good inhibitory effect on tumor growth. Realization of the strategy in vivo paves a new way to construct photothermal-triggered oxygen-independent therapeutic platform for clinical applications.

Simple fluorene oxadiazole-based Ir(iii) complexes with AIPE properties: synthesis, explosive detection and electroluminescence studies.

Two novel phosphorescent Ir(iii) complexes, Ir(fom)2(pic) and Ir(fof)2(pic), containing fluorene oxadiazole groups have been synthesized and characterized. The photophysical properties of the complexes have been investigated. Interestingly, both complexes exhibited aggregation-induced phosphorescent emission. The X-ray diffraction study showed that the AIPE properties resulted from weak π-π and C-HN hydrogen-bonding interactions in the aggregated state restricting the rotation of the phenyl groups in the cyclometalating ligands. Owing to the sensitive and selective luminescence quenching of the complexes using picric acid (PA), the complexes were used for PA detection in aqueous media. Additionally, electroluminescence devices have been fabricated using the complexes at 5%-30% doping concentrations. The devices based on Ir(fof)2(pic) obtained the highest luminance 11 877 cd m-2 and current efficiency 23.2 cd A-1, which implied that the incorporation of fluorine could improve the electron affinity and ameliorate the capability of electron injection or transporting.

Rational Design of Phosphorescent Iridium(III) Complexes for Selective Glutathione Sensing and Amplified Photodynamic Therapy.

It is a huge challenge to avoid irreversible damage to normal tissues during irradiation in photodynamic therapy (PDT) for cancer. An effective strategy is to develop smart photosensitizers, which exhibit amplified generation of reactive oxygen species (ROS) through triggering specific reaction in the tumor microenvironment. In this work, we designed a class of glutathione (GSH)-activatable photosensitizers (Ir1 and Ir4) based on an effective strategy of GSH-induced nucleophilic substitution reaction. The addition of GSH, induced changes in both phosphorescence intensity and lifetime of photosensitizers with high sensitivity. Importantly, the amount of singlet oxygen generated was increased significantly by GSH-induced activation reaction. Hence, the photosensitizers can selectively distinguish cancer cells from normal cells through luminescence and lifetime imaging, and can amplify PDT effects in cancer cells, owing to the evidently higher level of GSH compared to normal cells. This work presents a novel paradigm for GSH-amplified PDT against cancer cells and provides a new avenue for smart-responsive theranostic systems that can avoid nonspecific damage to normal cells.

Phosphorescent iridium(iii) complexes capable of imaging and distinguishing between exogenous and endogenous analytes in living cells.

Many luminescent probes have been developed for intracellular imaging and sensing. During cellular luminescence sensing, it is difficult to distinguish species generated inside cells from those internalized from extracellular environments since they are chemically the same and lead to the same luminescence response of the probes. Considering that endogenous species usually give more information about the physiological and pathological parameters of the cells while internalized species often reflect the extracellular environmental conditions, we herein reported a series of cyclometalated iridium(iii) complexes as phosphorescent probes that are partially retained in the cell membrane during their cellular uptake. The utilization of the probes for sensing and distinguishing between exogenous and endogenous analytes has been demonstrated using hypoxia and hypochlorite as two examples of target analytes. The endogenous analytes lead to the luminescence response of the intracellular probes while the exogenous analytes are reported by the probes retained in the cell membrane during their internalization.

Using Ultrafast Responsive Phosphorescent Nanoprobe to Visualize Elevated Peroxynitrite In Vitro and In Vivo via Ratiometric and Time-Resolved Photoluminescence Imaging.

Peroxynitrite (ONOO- ), a potent biological oxidant, which has a short half-life in physiological conditions, is related to many diseases. Accurate peroxynitrite determination with superior selectivity and sensitivity is important for understanding biological roles of peroxynitrite in different health and disease tissues. Autofluorescence is an inevitable interference in luminescence biodetection and bioimaging, which often reduces signal-to-noise ratio during detection. In this work, a phosphorescent peroxynitrite nanoprobe (MSN-ONOO) which displays two emission bands is prepared by immobilizing two long-lived phosphorescent iridium(III) complexes that are peroxynitrite-activable and -inert, respectively, into water-dispersible mesoporous silica nanoparticles. Owing to the fast response rate, excellent sensitivity and outstanding selectivity of the nanoprobe toward peroxynitrite, it is further used for peroxynitrite determination in vitro and in vivo via ratiometric photoluminescence imaging. More notably, taking advantage of the long-lived phosphorescence of MSN-ONOO, in vivo elevated peroxynitrite is imaged with diminished autofluorescence interference and improved signal-to-noise ratio via time-resolved photoluminescence imaging. As far as it is known, this is the first time for endogenous peroxynitrite detection in vivo via the time-resolved photoluminescence imaging. Furthermore, the production of peroxynitrite in inflamed tissues is visualized.

Dual-Phosphorescent Iridium(III) Complexes Extending Oxygen Sensing from Hypoxia to Hyperoxia.

Hypoxia and hyperoxia, referring to states of biological tissues in which oxygen supply is in sufficient and excessive, respectively, are often pathological conditions. Many luminescent oxygen probes have been developed for imaging intracellular and in vivo hypoxia, but their sensitivity toward hyperoxia becomes very low. Here we report a series of iridium(III) complexes in which limited internal conversion between two excited states results in dual phosphorescence from two different excited states upon excitation at a single wavelength. Structural manipulation of the complexes allows rational tuning of the dual-phosphorescence properties and the spectral profile response of the complexes toward oxygen. By manipulating the efficiency of internal conversion between the two emissive states, we obtained a complex exhibiting naked-eye distinguishable green, orange, and red emission in aqueous buffer solution under an atmosphere of N2, air, and O2, respectively. This complex is used for intracellular and in vivo oxygen sensing not only in the hypoxic region but also in normoxic and hyperoxic intervals. To the best of our knowledge, this is the first example of using a molecular probe for simultaneous bioimaging of hypoxia and hyperoxia.

Dual-Emissive Phosphorescent Polymer Probe for Accurate Temperature Sensing in Living Cells and Zebrafish Using Ratiometric and Phosphorescence Lifetime Imaging Microscopy.

Temperature plays an important part in many biochemical processes. Accurate diagnosis and proper treatment usually depend on precise measurement of temperature. In this work, a dual-emissive phosphorescent polymer temperature probe, composed of iridium(III) complexes as temperature sensitive unit with phosphorescence lifetime of ∼500 ns and europium(III) complexes as reference unit with lifetime of ∼400 µs, has been rationally designed and synthesized. Upon the increase of the temperature, the luminescence intensity from the iridium(III) complexes is enhanced, while that from the europium(III) complexes remains unchanged, which makes it possible for the ratiometric detection of temperature. Furthermore, the polymer also displays a significant change in emission lifetime accompanied by the temperature variation. By utilizing the laser scanning confocal microscope and time-resolved luminescence imaging systems, ratiometric and time-resolved luminescence imaging in Hela cells and zebrafish have been carried out. Notably, the intensity ratio and long-lifetime-based imaging can offer higher sensitivity, decrease the detection limit, and minimize the background interference from biosamples.

Achieving efficient photodynamic therapy under both normoxia and hypoxia using cyclometalated Ru(ii) photosensitizer through type I photochemical process.

Photodynamic therapy (PDT) through the generation of singlet oxygen utilizing photosensitizers (PSs) is significantly limited under hypoxic conditions in solid tumors. So it is meaningful to develop effective PSs which can maintain excellent therapeutic effects under hypoxia. Here we reported a coumarin-modified cyclometalated Ru(ii) photosensitizer (Ru2), which exhibits lower oxidation potential and stronger absorption in the visible region than the coumarin-free counterpart. The evaluation of the PDT effect was performed under both normoxia and hypoxia. The results showed that Ru2 has a better therapeutic effect than the coumarin-free counterpart in in vitro experiments. Especially under hypoxia, Ru2 still retained an excellent PDT effect, which can be attributed to the direct charge transfer between the excited PS and an adjacent substrate through a type I photochemical process, forming highly-oxidative hydroxyl radicals to damage tumor cells. The anti-tumor activity of Ru2 was further proven to be effective in tumor-bearing mice, and tumor growth was inhibited remarkably under PDT treatment.