Polypyridyl Complexes Research Articles

On the use of vibronic coherence to identify reaction coordinates for ultrafast excited-state dynamics of transition metal-based chromophores.

The question of whether one can use information from quantum coherence as a means of identifying vibrational degrees of freedom that are active along an excited-state reaction coordinate is discussed. Specifically, we are exploring the notion of whether quantum oscillations observed in single-wavelength kinetics data exhibiting coherence dephasing times that are intermediate between that expected for either pure electronic or pure vibrational dephasing are vibronic in nature and therefore may be coupled to electronic state-to-state evolution. In the case of a previously published Fe(II) polypyridyl complex, coherences observed subsequent to 1A1 → 1MLCT excitation were linked to large-amplitude motion of a portion of the ligand framework; dephasing times on the order of 200-300 fs suggested that these degrees of freedom could be associated with ultrafast (∼100 fs) conversion from the initially formed MLCT excited state to lower-energy, metal-centered ligand-field excited state(s) of the compound. Incorporation of an electronically benign but sterically restrictive Cu(I) ion into the superstructure designed to interfere with this motion yielded a compound exhibiting a ∼25-fold increase in the compound's MLCT lifetime, a result that was interpreted as confirmation of the initial hypothesis. However, new data acquired on a different chemical system - Cr(acac')3 (where acac' represents various derivatives of acetylacetonate) - yielded results that call into question this same hypothesis. Coherences observed subsequent to 4A2 → 4T2 ligand-field excitation on a series of molecules implicated similar vibrational degrees of freedom across the series, but exhibited dephasing times ranging from 340 fs to 2.5 ps without any clear correlation to the dynamics of excited-state evolution in the system. Taken together, the results obtained on both of these chemical platforms suggest that while identification of coherences can indeed point to degrees of freedom that should be considered as candidate modes for defining reaction trajectories, our understanding of the factors that determine the interplay across coherences, dephasing times, and electronic and geometric structure is insufficient at the present time to view this parameter as a robust metric for differentiating active versus spectator modes for ultrafast dynamics.

Increasing the cytotoxicity of Ru(II) polypyridyl complexes by tuning the electron-donating ability of 1,10-phenanthroline ligands.

Ruthenium (Ru)-based chemotherapeutic agents are a choice to replace traditional platinum-containing metallodrugs due to fewer side effects. It has been proved that the mechanism of Ru complex drugs is to highly likely bind with DNA and certain proteins, which also highly depends on the electronic structures of Ru complexes. However, the relationship between electronic properties and chemotherapeutic activities has not yet been completely systemically investigated, which limits the effective drug design strategies. Herein, we propose that increasing the electron densities of Ru would enhance the nucleophilic substitution rate of chlorine atoms (Cl) on Ru, providing better bioactivity against both amino acids and nucleic acids. A series of complexes with various optimized electron-donating groups (EDGs) were synthesized according to DFT calculations. In addition, kinetics substitution with L-histidine, DNA binding experiments, and cell cytotoxicity studies verified our assumptions. Surprisingly, these complexes could also be potential cellular imaging probes via an unprecedented "off-on" light-switching mechanism of living cells, which was caused by the "HOMO-LUMO" distribution changes of Ru complexes after interaction with DNA. Accordingly, the reactivity and selectivity demonstrated by these compounds support the further development of these Ru complexes in cancer treatments and afford strategic perspectives on the development of metal complexes as chemotherapeutic agents and bioimaging probes.

Mononuclear Co(II) polypyridyl complexes: synthesis, molecular structure, DNA binding/cleavage, radical scavenging, docking studies and anticancer activities.

Mononuclear Co(II) complexes [CoII(L)Cl2]; 1, [CoII(L)(bpy)Cl]PF6; 2, [CoII(L)(phen)Cl]PF6; 3 and [CoII(L)(pic)Cl]; 4, (where L = N,N-bis(pyridin-2-ylmethyl)aniline, bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, pic = picolinic acid) were systematically synthesized and characterized by different analytical and spectroscopic methods. All the complexes were structurally identified by single-crystal X-ray diffraction analysis. Penta-coordinated complex 1 adopted a distorted trigonal bipyramidal geometry, whereas hexacoordinated complexes 2-4 have distorted octahedral geometry. The interactions of salmon sperm DNA (ss-DNA) with our synthesized complexes 1-4 were investigated by absorbance and fluorescence spectroscopy. All the complexes are very susceptible to DNA binding and exhibited binding affinities (Kb) in the order of ∼104 M-1, indicating their strong interaction with ss-DNA. The Stern-Volmer constant (Ksv) ranged from 0.46 ± 0.01 ×104 to 1.08 ± 0.04 × 104 M-1, suggesting weak or moderate binding with DNA. Agarose gel electrophoresis revealed the DNA cleavage activity in vitro for 2-4, which could efficiently cleave the supercoiled plasmid DNA without any external agents; however, with the addition of H2O2, the cleavage property was enhanced. Live-cell imaging and other biochemical assays demonstrated the ability of Co(II) complexes 1-4 to induce significant cytotoxicity in A549 lung cancer cells with IC50 values of 32.14 ± 1.3 μM, 3.14 ± 0.16 μM, 15.78 ± 0.72 μM and 18.45 ± 0.92 μM, and in MDA-MB-231 breast cancer cells with IC50 values of 20.42 ± 0.92 μM, 0.41 ± 0.02 μM, 2.31 ± 0.12 μM and 9.67 ± 0.35 μM, respectively.

Balancing the interplay between ligand ejection and therapeutic window light absorption in ruthenium polypyridyl complexes.

Ruthenium polypyridyl complexes have gained significant interest as photochemotherapies (PCTs) where their excited-state properties play a critical role in the photo-cytotoxicity mechanism and efficacy. Herein we report a systematic electrochemical, spectrochemical, and photophysical analysis of a series of ruthenium(II) polypyridyl complexes of the type [Ru(bpy)2(N-N)]2+ (where bpy = 2,2'-bipyridine; N-N is a bidentate polypyridyl ligand) designed to mimic PCTs. In this series, the N-N ligand was modified through increased conjugation and/or incorporation of electronegative heteroatoms to shift the metal-to-ligand charge-transfer (MLCT) absorptions near the therapeutic window for PCTs (600-1100 nm) while incorporating steric bulk to trigger photoinduced ligand dissociation. The lowest energy MLCT absorptions were red-shifted from λmax = 454 nm to 564 nm, with emission energies decreasing from λmax = 620 nm to 850 nm. Photoinduced ligand ejection and temperature-dependent emission studies revealed an important interplay between red-shifting MLCT absorptions and accessing the dissociative 3dd* states, with energy barriers between the 3MLCT* and 3dd* states ranging from 850 cm-1 to 2580 cm-1 for the complexes measured. This work demonstrates the importance of understanding both the MLCT manifold and 3dd* state energy levels in the future design of ligands and complexes for PCT.

Ruthenium photosensitizer anchored gold nanorods for synergistic photodynamic and photothermal therapy.

Ruthenium polypyridyl complexes have been widely used as bioprobes and photosensitizers. However, several disadvantages including slow cellular uptake, nonspecific binding with biomolecules and toxicity limit their applications. In this study, a nanocarrier of human serum albumin coated gold nanorods was developed to deliver a ruthenium photosensitizer for PDT/PTT combination therapy. The HSA coating endowed the nanodrug with high biocompatibility and stability under physiological conditions. Ru-GNR-HSANPs generate 1O2 and hydroxyl radicals to kill cancer cells under blue light irradiation, and exhibit excellent photothermal anticancer effects under 808 nm light irradiation. Significant synergistic anticancer effects were achieved by combined PDT/PTT therapy. Importantly, Ru-GNR-HSANPs can have the synergistic PDT/PTT functions with no need of drug release from the carrier.

New ruthenium polypyridyl complexes as potential sensors of acetonitrile and catalysts for water oxidation

New ruthenium(ii) polypyridyl complexes of formulae [RuCl(Me2Ntrpy)(bpy-OMe)]Cl, 1, and [Ru(Me2Ntrpy)(bpy-OMe)(OH2)](CF3SO3)2, 2, with Me2Ntrpy = 4′-N,N-dimethylamino-2,2′:6′,2′′-terpyridine and bpy-OMe = 4,4′-dimethoxy-2,2′-bipyridine, were synthetized and characterized by spectroscopic and electrochemical techniques. Besides, [Ru(Me2Ntrpy)(bpy-OMe)(NCCH3)]2+, 3, was obtained and characterized by UV-vis spectroscopy in acetonitrile solution. All experimental results were complemented with DFT and TD-DFT calculations. The complete structure of complex 1 was determined by X-ray diffraction, evidencing that the Ru–N and Ru–Cl bond lengths are longer than those determined in [RuCl(trpy)(bpy)](PF6). The strong electron donating properties of the substituents of both bpy and trpy rings in complexes 1 and 2 led to their potential applications for detecting traces of acetonitrile as a contaminant in aqueous solutions of radiopharmaceuticals and to utilization of complex 2 as a promising candidate for catalyzing water oxidation processes.

Visible light-driven CO2 reduction with a Ru polypyridyl complex bearing an N-heterocyclic carbene moiety.

A novel Ru polypyridyl complex with an N-heterocyclic carbene ligand was successfully synthesised and characterised. The complex exhibited an intense absorption band in the visible-light region derived from the strong electron-donating character of the carbene ligand, and efficiently catalysed the visible light-driven CO2 reduction with the reaction rate of 36.7 h-1.

The Use of Ruthenium Complexes as Molecular Probes for Non-Canonical DNA

This study considered the preparation of a new DNA binding Ruthenium polypyridyl complex possessing an infrared active nitrile group. The binding abilities of a novel Ruthenium complex, [Ru(TMP)2DPPZ-10-CN], to various forms of DNA—both canonical and non-canonical—were examined by performing multiple DNA titrations. DNA is of great interest as it is the carrier of genetic information for all living things. Damage to DNA can have drastically detrimental effects, so the study of its structure and replication is of great importance. Two non-canonical structures that are important are the G-quadruplex and i-motif which form at the telomeric and regulatory regions of genes, respectively, and have the ability to block telomerase activity and influence transcription. The complex was synthesized by microwave irradiation and purified using a silica column and an ion exchange with Amberlite 402. Six titrations were, then, performed with salmon sperm dsDNA, guanine monophosphate (GMP), G4T4G4, human telomere G-quadruplex, i-motif C5T3, and i-motif C30. The complex was found to favor non-canonical structures, particularly the G-quadruplex structure, because of its high [bp]/[Ru] concentrations. The higher concentration of base pairs or structures per Ruthenium molecule indicated that the complex had a high binding affinity for that particular DNA structure. These results support the notion that Ruthenium metal complexes can be used for theragnostic purposes and can be used to target the telomeric region of genes where G-quadruplex structures can be found and influence transcription initiation and inhibit telomerase activity.

The Use of Ruthenium Complexes as Molecular Probes for Non-Canonical DNA” by Eleni Sullivan

This study considered the preparation of a new DNA binding Ruthenium polypyridyl complex possessing an infrared active nitrile group. The binding abilities of a novel Ruthenium complex, [Ru(TMP)2DPPZ-10-CN], to various forms of DNA—both canonical and non-canonical—were examined by performing multiple DNA titrations. DNA is of great interest as it is the carrier of genetic information for all living things. Damage to DNA can have drastically detrimental effects, so the study of its structure and replication is of great importance. Two non-canonical structures that are important are the G-quadruplex and i-motif which form at the telomeric and regulatory regions of genes, respectively, and have the ability to block telomerase activity and influence transcription. The complex was synthesized by microwave irradiation and purified using a silica column and an ion exchange with Amberlite 402. Six titrations were, then, performed with salmon sperm dsDNA, guanine monophosphate (GMP), G4T4G4, human telomere G-quadruplex, i-motif C5T3, and i-motif C30. The complex was found to favor non-canonical structures, particularly the G-quadruplex structure, because of its high [bp]/[Ru] concentrations. The higher concentration of base pairs or structures per Ruthenium molecule indicated that the complex had a high binding affinity for that particular DNA structure. These results support the notion that Ruthenium metal complexes can be used for theragnostic purposes and can be used to target the telomeric region of genes where G-quadruplex structures can be found and influence transcription initiation and inhibit telomerase activity.

Ligand-Field Effects in a Ruthenium(II) Polypyridyl Complex Probed by Femtosecond X-ray Absorption Spectroscopy.

We employ femtosecond X-ray absorption spectroscopy of [Ru(m-bpy)3]2+ (m-bpy = 6-methyl-2,2'-bipyridine) to elucidate the time evolution of the spin and charge density upon metal-to-ligand charge-transfer (MLCT) excitation. The core-level transitions at the Ru L3-edge reveal a very short MLCT lifetime of 0.9 ps and relaxation to the lowest triplet metal-centered state (3MC) which exhibits a lifetime of about 300 ps. Time-dependent density functional theory relates ligand methylation to a lower ligand field strength that stabilizes the 3MC state. A quarter of the 3MLCT population appears to be trapped which may be attributed to intramolecular vibrational relaxation or further electron transfer to the solvent. Our results demonstrate that small changes in the ligand field allow control of the photophysical properties. Moreover, this study underscores the high information content of femtosecond L-edge spectroscopy as a probe of valence charge density and spin-state in 4d transition metals.

Solution-Based Self-Assembly and Stability of Ruthenium(II) Tris-bipyridyl Monolayers on Gold.

Ruthenium(II) polypyridyl complexes are commonly applied as photosensitizers in the fields of artificial photosynthesis and light harvesting. Their immobilization on gold surfaces is also of interest for sensing and biological applications. Here, we report the self-assembly of [Ru(dmbpy)2(dcbpy)](PF6)2 complexes on gold substrates from solution (dmbpy: 4,4'-dimethyl-2,2'-bipyridine; dcbpy: 2,2'-bipyridine-4,4'-dicarboxylic acid). Applying X-ray photoelectron spectroscopy, we demonstrate the formation of self-assembled monolayers (SAMs) of the Ru(II) complexes upon loss of counterions with carboxylate groups oriented toward the gold surface. We investigate the stability of the formed SAMs toward the substitution in solvents with competing aliphatic and aromatic thiols such as 4'-nitro[1,1'-biphenyl]-4-thiol, [1,1'-biphenyl]-4-thiol, and 1-hexadecanethiol. We show that the exchange reactions may lead to both complete replacement of the Ru(II) complexes and controlled formation of mixed SAMs. Moreover, we demonstrate that thiol-based SAMs can also be replaced completely from gold via their immersion into solutions of [Ru(dmbpy)2(dcbpy)](PF6)2, indicating a relatively high stability for the Ru(II) complex SAMs. Our findings open up a variety of opportunities for applications of carboxylate-based SAMs on gold in nanotechnology.

Electrochromism or Water Splitting in Neutral Aqueous Solutions by a Metallo‐Organic Assembly

Herein, two different functionalities of one metallo‐organic film assembled on the surface of a transparent metal‐oxide electrode are described. Two redox‐active elements, electrochromic iron–polypyridyl complexes and catalytically active palladium centers, operate by applying different potentials in aqueous solutions. The color of the material can be cycled 1500 times from dark purple to colorless by electrochemically addressing the Fe2+/3+ centers at 0.0–1.0 V (vs Ag/Ag+). The differences between the transmittance of these two states is high: ΔT = 52%. Catalytic water oxidation can occur by palladium oxide particles that form in situ by applying a higher potential (1.22–2.0 V vs Ag/Ag+), resulting in the formation of hydrogen and oxygen. The catalyst is active for at least 7 h in an aqueous electrolyte at pH = 6.9, with a Faradaic efficiency of ≈70%.

Evaluating the mechanisms of action and subcellular localization of ruthenium(II)-based photosensitizers.

The identification of ruthenium(II) polypyridyl complexes as photosensitizers in photodynamic therapy (PDT) for the treatment of cancer is progressing rapidly. Due to their favorable photophysical and photochemical properties, Ru(II)-based photosensitizers have absorption in the visible spectrum, can be irradiated via one- and two-photon excitation within the PDT window, and yield potent oxygen-dependent and/or oxygen-independent photobiological activities. Herein, we present a current overview of the mechanisms of action and subcellular localization of Ru(II)-based photosensitizers in the treatment of cancer. These photosensitizers are highlighted from a medicinal chemistry and chemical biology perspective. However, although this field is burgeoning, challenges and limitations remain in the photosensitization strategies and clinical translation.

Ru(II)-Based Acetylacetonate Complexes Induce Apoptosis Selectively in Cancer Cells.

The synthesis, chemical and biological characterization of seven Ru(II) polypyridyl complexes containing acetylacetonate (acac) ligands are reported. Electronic absorption spectra were determined and electrochemical potentials consistent with Ru(III/II) couples ranging from +0.60 to +0.73 V vs Ag/AgCl were measured. A series of complexes were screened against MDA-MB-231, DU-145, and MCF-10A cell lines to evaluate their cytotoxicities in cancer and normal cell lines. Although most complexes were either nontoxic or equipotent in cancer cells and normal cell lines, compound 1, [Ru(dpqy)(acac)(py)](PF6), where dqpy is 2,6-di(quinolin-2-yl)pyridine, showed up to 2.5:1.0 selectivity for cancer as compared to normal cells, along with nanomolar EC50 values in MDA-MB-231 cells. Lipophilicity, determined as the octanol/water partition coefficient, log Po/w, ranged from -0.33 (0.06) to 1.15 (0.10) for the complexes. Although cytotoxicity was not correlated with electrochemical potentials, a moderate linear correlation between lipophilicity and toxicities was observed. Cell death mechanism studies indicated that several of the Ru-acac compounds, including 1, induce apoptosis in MDA-MB-231 cells.

Tetrazole-Substituted isomeric ruthenium polypyridyl complexes for low overpotential electrocatalytic CO2 reduction

Introducing tetrazole moiety to the ligand framework of two isomeric ruthenium catalysts, cis/trans-[Ru(tpy)(mtzp)(CH3CN)]2+ (tpy = 2,2′:6′,2′'-terpyridine, mtzp = 2-(1-methyl-1H-tetrazol-5-yl)pyridine), for the electrochemical reduction of CO2 to CO has altered the catalytic pathway with significantly low overpotential (0.37 V) compared to its analogous catalysts. Without manipulating steric effects, only the electronic nature of tetrazole moiety enables CO2 binding to ruthenium center to form metallocarboxylate intermediate just after one-electron reduction. This is the first synthesized isomeric pair of ruthenium complex follow ECE (E = electron transfer, C = chemical reaction) mechanism for electrocatalytic reduction of CO2. By successful characterization of the Ru–CO intermediate with the help of 13C NMR, spectro-electrochemical studies and analysis of byproducts formed during the electrocatalysis, a mechanism of CO2 reduction has been established in presence of water and anhydrous conditions which is further supported by density functional theory (DFT).

Nitroimidazole-Based Ruthenium(II) Complexes: Playing with Structural Parameters to Design Photostable and Light-Responsive Antibacterial Agents.

5-Nitroimidazole (5NIMH), chosen as a molecular model of nitroimidazole derivatives, which represent a broad-spectrum class of antimicrobials, was incorporated into the ruthenium complexes [Ru(tpy)(phen)(5NIM)]PF6 (1) and [Ru(tpy)(dmp)(5NIM)]PF6 (2) (tpy = terpyridine, phen = phenanthroline, dmp = 2,9-dimethyl-1,10-phenanthroline). Besides the uncommon metal coordination of 5-nitroimidazole in its imidazolate form (5NIM), the different architectures of the spectator ligands (phen and dmp) were exploited to tune the "mode of action" of the resulting complexes, passing from a photostable compound where the redox properties of 5NIMH are preserved (1) to one suitable for the nitroimidazole phototriggered release (2) and whose antibacterial activity against B. subtilis, chosen as cellular model, is effectively improved upon light exposure. This study may provide a fundamental knowledge on the use of Ru(II)-polypyridyl complexes to incorporate and/or photorelease biologically relevant nitroimidazole derivatives in the design of a novel class of antimicrobials.

Materials for optical oxygen sensing under high hydrostatic pressure

Optical oxygen sensors based on indicators immobilized into porous and nonporous matrix materials were investigated in regard to their sensing behaviour under high hydrostatic pressure. The sensors were subjected to step-wise pressure increase up to approximately 200 bar in multiple cycles using a custom-made chamber. The investigated materials are based on oxygen indicators (a platinum(II) benzoporphyrin dye and ruthenium(II) polypyridyl complexes) immobilized in microparticles: silica gels of different porosities, controlled pore glass, poly(phenylsilsesquioxane), crosslinked polystyrene, the metal-organic frameworks ZIF-8 and UiO-66. The microparticles are in turn dispersed in highly oxygen-permeable silicone and Hyflon AD matrices. Homogeneous films of dye doped polystyrene and polyurethane hydrogels as well as non-porous polystyrene nanospheres directly dispersed in water were used for comparison purposes. All the porous materials were found to be stable under elevated hydrostatic pressure. Although the actual oxygen concentration remained unchanged upon increasing pressure, the sensors demonstrated an apparent decrease of calculated oxygen concentration (between −0.02 and −0.45 mg O2 L-1 H2O per 100 bar), which was fully reversible. Furthermore, the kinetics of the pressure response of the sensors was determined in experiments with high temporal resolution. Spikes attributed to pressure-induced oxygen diffusion between sensor components were observed within the first seconds after pressurization/depressurization.

Covalent Grafting of Ruthenium Complexes on Iron Oxide Nanoparticles: Hybrid Materials for Photocatalytic Water Oxidation.

The present environmental crisis prompts the search for renewable energy sources such as solar-driven production of hydrogen from water. Herein, we report an efficient hybrid photocatalyst for water oxidation, consisting of a ruthenium polypyridyl complex covalently grafted on core/shell Fe@FeOx nanoparticles via a phosphonic acid group. The photoelectrochemical measurements were performed under 1 sun illumination in 1 M KOH. The photocurrent density of this hybrid photoanode reached 20 μA/cm2 (applied potential of +1.0 V vs reversible hydrogen electrode), corresponding to a turnover frequency of 0.02 s-1. This performance represents a 9-fold enhancement of that achieved with a mixture of Fe@FeOx nanoparticles and a linker-free ruthenium polypyridyl photosensitizer. This increase in performance could be attributed to a more efficient electron transfer between the ruthenium photosensitizer and the Fe@FeOx catalyst as a consequence of the covalent link between these two species through the phosphonate pendant group.

Recent Approaches towards the Development of Ru(II) Polypyridyl Complexes for Anticancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a remarkable alternative or complementary technique to chemotherapy, radiotherapy or immunotherapy to treat certain forms of cancer. The synergistic effect of light, photosensitizer (PS) and oxygen allows for the treatment of tumours with an extremely high spatio-tumoral control, therefore minimizing the severe side effects usually observed in chemotherapy. The currently employed PDT PSs based on porphyrins have, in some cases, some limitations, which include a low absorbance in the therapeutic window, a low body clearance, photobleaching, among others. In this context, Ru(ii) polypyridyl complexes are interesting alternatives. They have low lying excited energy states and the presence of a heavy metal increases the possibility of spin-orbit coupling. Moreover, their photophysical properties are relatively easy to tune and they have very low photobleaching rates. All of these make them attractive candidates for further development as therapeutically suitable PDT PSs. In this review, after having presented this field of research, we discuss the developments made by our group in this field of research since 2017. We notably describe how we tuned the photophysical properties of our complexes from the visible region to the therapeutically suitable red region. This was accompanied by the preparation of PSs with enhanced phototoxicity and high phototoxicity index. We also discuss the use of two-photon excitation to eradicate tumours in nude mice. Furthermore, we describe our approach for the selective delivery of our complexes using targeting agents. Lastly, we report on our very recent synergistic approach to treat cancer using bimetallic Ru(ii)-Pt(iv) prodrug candidates.

Local DNA microviscosity converts ruthenium polypyridyl complexes to ultrasensitive photosensitizers

In this manuscript, we have reported how DNA binding of ruthenium polypyridyl complexes (RPCs) make them more efficient photosensitizer. The DNA binding of RPCs results in a significant enhancement of their emission intensity and excited-state lifetime. As a result, upon visible-light irradiation, these complexes generate reactive singlet oxygen, which causes selective DNA damage and kills cancer cells. This investigation also demonstrates the effect of light-driven RPCs on bacterial growth arrest through DNA nicking and differential localization in cancer and non-cancer cells. The local DNA microviscosity suppresses the non-radiative pathways which causes a large enhancement in the emission intensity and the excited state lifetime. The visible-light-triggered singlet-oxygen efficiently produces nick in DNA and inhibits bacterial growth. RPCs also localize inside the cancer cell nucleus and in the vicinity of the nuclear membrane of non-cancerous cells, confirmed by live-cell confocal microscopy. The results provide a facile platform for the novel antibiotic intended discovery combined with cancer therapy.