Intraligand Excited States Turn a Ruthenium Oligothiophene Complex into a Light-Triggered Ubertoxin with Anticancer Effects in Extreme Hypoxia.

Ru(II) complexes that undergo photosubstitution reactions from triplet metal-centered (3MC) excited states are of interest in photochemotherapy (PCT) due to their potential to produce cytotoxic effects in hypoxia. Dual-action systems that incorporate this stoichiometric mode to complement the oxygen-dependent photosensitization pathways that define photodynamic therapy (PDT) are poised to maintain antitumor activity regardless of the oxygenation status. Herein, we examine the way in which these two pathways influence photocytotoxicity in normoxia and in hypoxia using the [Ru(dmp)2(IP-nT)]2+ series (where dmp = 2,9-dimethyl-1,10-phenanthroline and IP-nT = imidazo[4,5-f][1,10]phenanthroline tethered to n = 0-4 thiophene rings) to switch the dominant excited state from the metal-based 3MC state in the case of Ru-phen-Ru-1T to the ligand-based 3ILCT state for Ru-3T and Ru-4T. Ru-phen-Ru-1T, having dominant 3MC states and the largest photosubstitution quantum yields, are inactive in both normoxia and hypoxia. Ru-3T and Ru-4T, with dominant 3IL/3ILCT states and long triplet lifetimes (τTA = 20-25 µs), have the poorest photosubstitution quantum yields, yet are extremely active. In the best instances, Ru-4T exhibit attomolar phototoxicity toward SKMEL28 cells in normoxia and picomolar in hypoxia, with phototherapeutic index values in normoxia of 105-1012 and 103-106 in hypoxia. While maximizing excited-state deactivation through photodissociative 3MC states did not result in bonafide dual-action PDT/PCT agents, the study has produced the most potent photosensitizer we know of to date. The extraordinary photosensitizing capacity of Ru-3T and Ru-4T may stem from a combination of very efficient 1O2 production and possibly complementary type I pathways via 3ILCT excited states.

Interaction with a Biomolecule Facilitates the Formation of the Function-Determining Long-Lived Triplet State in a Ruthenium Complex for Photodynamic Therapy.

TLD1433 is the first ruthenium (Ru)-based photodynamic therapy (PDT) agent to advance to clinical trials and is currently in a phase II study for treating nonmuscle bladder cancer with PDT. Herein, we present a photophysical study of TLD1433 and its derivative TLD1633 using complex, biologically relevant solvents to elucidate the excited-state properties that are key for biological activity. The complexes incorporate an imidazo [4,5-f][1,10]phenanthroline (IP) ligand appended to α-ter- or quaterthiophene, respectively, where TLD1433 = [Ru(4,4'-dmb)2(IP-3T)]Cl2 and TLD1633 = [Ru(4,4'-dmb)2(IP-4T)]Cl2 (4,4'-dmb = 4,4'-dimethyl-2,2'-bipyridine; 3T = α-terthiophene; 4T = α-quaterthiophene). Time-resolved transient absorption experiments demonstrate that the excited-state dynamics of the complexes change upon interaction with biological macromolecules (e.g., DNA). In this case, the accessibility of the lowest-energy triplet intraligand charge-transfer (3ILCT) state (T1) is increased at the expense of a higher-lying 3ILCT state. We attribute this behavior to the increased rigidity of the ligand framework upon binding to DNA, which prolongs the lifetime of the T1 state. This lowest-lying state is primarily responsible for O2 sensitization and hence photoinduced cytotoxicity. Therefore, to gain a realistic picture of the excited-state kinetics that underlie the photoinduced function of the complexes, it is necessary to interrogate their photophysical dynamics in the presence of biological targets once they are known.

Photophysical Properties and Photobiological Activities of Ruthenium(II) Complexes Bearing π-Expansive Cyclometalating Ligands with Thienyl Groups.

A new family of cyclometalated ruthenium(II) complexes [Ru(N^N)2(C^N)]+ derived from the π-extended benzo[h]imidazo[4,5-f]quinolone ligand appended with thienyl groups (n = 1-4, compounds 1-4) was prepared and its members were characterized for their chemical, photophysical, and photobiological properties. The lipophilicities of 1-4, determined as octanol-water partition coefficients (log Po/w), were positive and increased with the number of thienyl units. The absorption and emission bands of the C^N compounds were red-shifted by up to 200 nm relative to the analogous Ru(II) diimine systems. All of the complexes exhibited dual emission with the intraligand fluorescence (1IL, C^N-based) shifting to lower energies with increasing n and the metal-to-ligand charge transfer phosphorescence (3MLCT, N^N-based) remaining unchanged. Compounds 1-3 exhibited excited state absorption (ESA) profiles consistent with lowest-lying 3MLCT states when probed by nanosecond transient absorption (TA) spectroscopy with 532 nm excitation and had contributions from 1IL(C^N) states with 355 nm excitation. These assignments were supported by the lifetimes observed (<10 ns for the 1IL states and around 20 ns for the 3MLCT states) as well as a noticeable ESA for 3 with 355 nm excitation that did not occur with 532 nm excitation. Compound 4 was the only member of the family with two 3MLCT emissive lifetimes (15, 110 ns), and the TA spectra collected with both 355 and 532 nm excitation was assigned to the 3IL state, which was corroborated by its 4-6 µs lifetime. The ESA for 4 had a rise time of approximately 10 ns and an initial decay of 110 ns, which suggests a possible 3MLCT-3IL excited state equilibrium that results in delayed emission from the 3MLCT state. Compound 4 was nontoxic toward human skin melanoma cells (SKMEL28) in the dark (EC50 = >300 µM); 1-3 were cytotoxic and yielded EC50 values between 1 and 20 µM. The photocytotoxicites with visible light ranged from 87 nM with a phototherapeutic index (PI) of 13 for 1 to approximately 1 µM (PI = >267) for 4. With red light, EC50 values varied from 270 nM (PI = 21) for 3 to 12 µM for 4 (PI = >25). The larger PIs for 4, especially with visible light, were attributed to the much lower dark cytotoxicity for this compound. Because the dark cytotoxicity contributes substantially to the observed photocytotoxicity for 1-3, it was not possible to assess whether the 3IL state of 4 led to a much more potent phototoxic mechanism in the absence of dark toxicity. There was no stark contrast in cellular uptake and accumulation by laser scanning confocal and differential interference contrast microscopy to explain the large differences in dark toxicities between 1-3 and 4. Nevertheless, the study highlights a new family of Ru(II) C^N complexes where π-conjugation beyond a certain point results in low dark cytotoxicity with high photocytotoxicity, opposing the notion that cyclometalated Ru(II) systems are too toxic to be phototherapeutic agents.

Photodynamic therapy of melanoma with new, structurally similar, NIR-absorbing ruthenium (II) complexes promotes tumor growth control via distinct hallmarks of immunogenic cell death.

Cancer therapies that generate T cell-based anti-cancer immune responses are critical for clinical success and are favored over traditional therapies. One way to elicit T cell immune responses and generate long-lasting anti-cancer immunity is through induction of immunogenic cell death (ICD), a form of regulated cell death that promotes antigenicity and adjuvanticity within dying cells. Therefore, research in the last decade has focused on developing cancer therapies which stimulate ICD. Herein, we report novel photodynamic therapy (PDT) compounds with immunomodulatory and ICD inducing properties. PDT is a clinically approved, minimally invasive anti-cancer treatment option and has been extensively investigated for its tumor-destroying properties, lower side effects, and immune activation capabilities. In this study, we explore two structurally related ruthenium compounds, ML19B01 and ML19B02, that can be activated with near infrared light to elicit superior cytotoxic properties. In addition to its direct cell killing abilities, we investigated the effect of our PSs on immunological pathways upon activation. PDT treatment with ML19B01 and ML19B02 induced differential expression of reactive oxygen species, proinflammatory response-mediating genes, and heat shock proteins. Dying melanoma cells induced by ML19B01-PDT and ML19B02-PDT contained ICD hallmarks such as calreticulin, ATP, and HMGB1, initiated activation of antigen presenting cells, and were efficiently phagocytosed by bone marrow-derived dendritic cells. Most importantly, despite the distinct profiles of ICD hallmark inducing capacities, vaccination with both PDT-induced dying cancer cells established anti-tumor immunity that protected mice against subsequent challenge with melanoma cells.

Near-infrared absorbing Ru(ii) complexes act as immunoprotective photodynamic therapy (PDT) agents against aggressive melanoma.

Mounting evidence over the past 20 years suggests that photodynamic therapy (PDT), an anticancer modality known mostly as a local treatment, has the capacity to invoke a systemic antitumor immune response, leading to protection against tumor recurrence. For aggressive cancers such as melanoma, where chemotherapy and radiotherapy are ineffective, immunomodulating PDT as an adjuvant to surgery is of interest. Towards the development of specialized photosensitizers (PSs) for treating pigmented melanomas, nine new near-infrared (NIR) absorbing PSs based on a Ru(ii) tris-heteroleptic scaffold [Ru(NNN)(NN)(L)]Cl n , were explored. Compounds 2, 6, and 9 exhibited high potency toward melanoma cells, with visible EC50 values as low as 0.292-0.602 µM and PIs as high as 156-360. Single-micromolar phototoxicity was obtained with NIR-light (733 nm) with PIs up to 71. The common feature of these lead NIR PSs was an accessible low-energy triplet intraligand (3IL) excited state for high singlet oxygen (1O2) quantum yields (69-93%), which was only possible when the photosensitizing 3IL states were lower in energy than the lowest triplet metal-to-ligand charge transfer (3MLCT) excited states that typically govern Ru(ii) polypyridyl photophysics. PDT treatment with 2 elicited a pro-inflammatory response alongside immunogenic cell death in mouse B16F10 melanoma cells and proved safe for in vivo administration (maximum tolerated dose = 50 mg kg-1). Female and male mice vaccinated with B16F10 cells that were PDT-treated with 2 and challenged with live B16F10 cells exhibited 80 and 55% protection from tumor growth, respectively, leading to significantly improved survival and excellent hazard ratios of ≤0.2.