Ru(II) Oligothienyl Complexes with Fluorinated Ligands: Photophysical, Electrochemical, and Photobiological Properties.

A series of Ru(II) complexes incorporating two 4,4'-bis(trifluoromethyl)-2,2'-bipyridine (4,4'-btfmb) coligands and thienyl-appended imidazo[4,5-f][1,10]phenanthroline (IP-nT) ligands was characterized and assessed for phototherapy effects toward cancer cells. The [Ru(4,4'-btfmb)2(IP-nT)]2+ scaffold has greater overall redox activity compared to Ru(II) polypyridyl complexes such as [Ru(bpy)3]2+. Ru-1T-Ru-4T have additional oxidations due to the nT group and additional reductions due to the 4,4'-btfmb ligands. Ru-2T-Ru-4T also exhibit nT-based reductions. Ru-4T exhibits two oxidations and eight reductions within the potential window of -3 to +1.5 V. The lowest-lying triplets (T1) for Ru-0T-2T are metal-to-ligand charge-transfer (3MLCT) excited states with lifetimes around 1 µs, whereas T1 for Ru-3T-4T is longer-lived (∼20-24 µs) and of significant intraligand charge-transfer (3ILCT) character. Phototoxicity toward melanoma cells (SK-MEL-28) increases with n, with Ru-4T having a visible EC50 value as low as 9 nM and PI as large as 12,000. Ru-3T and Ru-4T retain some of this activity in hypoxia, where Ru-4T has a visible EC50 as low as 35 nM and PI as high as 2900. Activity over six biological replicates is consistent and within an order of magnitude. These results demonstrate the importance of lowest-lying 3ILCT states for phototoxicity and maintaining activity in hypoxia.

Ru(II) Phenanthroline-Based Oligothienyl Complexes as Phototherapy Agents.

Ru(II) polypyridyl complexes have gained widespread attention as photosensitizers for photodynamic therapy (PDT). Herein, we systematically investigate a series of the type [Ru(phen)2(IP-nT)]2+, featuring 1,10-phenanthroline (phen) coligands and imidazo[4,5-f][1,10]phenanthroline ligands tethered to n = 0-4 thiophene rings (IP-nT). The complexes were characterized and investigated for their electrochemical, spectroscopic, and (photo)biological properties. The electrochemical oxidation of the nT unit shifted by -350 mV as n = 1 → 4 (+920 mV for Ru-1T, +570 mV for Ru-4T); nT reductions were observed in complexes Ru-3T (-2530 mV) and Ru-4T (-2300 mV). Singlet oxygen quantum yields ranged from 0.53 to 0.88, with Ru-3T and Ru-4T being equally efficient (∼0.88). Time-resolved absorption spectra of Ru-0T-1T were dominated by metal-to-ligand charge-transfer (3MLCT) states (τTA = 0.40-0.85 µs), but long-lived intraligand charge-transfer (3ILCT) states were observed in Ru-2T-4T (τTA = 25-148 µs). The 3ILCT energies of Ru-3T and Ru-4T were computed to be 1.6 and 1.4 eV, respectively. The phototherapeutic efficacy against melanoma cells (SK-MEL-28) under broad-band visible light (400-700 nm) increases as n = 0 → 4: Ru-0T was inactive up to 300 µM, Ru-1T-2T were moderately active (EC50 ∼ 600 nM, PI = 200), and Ru-3T (EC50 = 57 nM, PI > 1100) and Ru-4T (EC50 = 740 pM, PI = 114,000) were the most phototoxic. The activity diminishes with longer wavelengths of light and is completely suppressed for all complexes except Ru-3T and Ru-4T in hypoxia. Ru-4T is the more potent and robust PS in 1% O2 over seven biological replicates (avg EC50 = 1.3 µM, avg PI = 985). Ru-3T exhibited hypoxic activity in five of seven replicates, underscoring the need for biological replicates in compound evaluation. Singlet oxygen sensitization is likely responsible for phototoxic effects of the compounds in normoxia, but the presence of redox-active excited states may facilitate additional photoactive pathways for complexes with three or more thienyl groups. The 3ILCT state with its extended lifetime (30-40× longer than the 3MLCT state for Ru-3T and Ru-4T) implicates its predominant role in photocytotoxicity.

High quantum efficiency ruthenium coordination complex photosensitizer for improved radiation-activated Photodynamic Therapy.

Traditional external light-based Photodynamic Therapy (PDT)'s application is limited to the surface and minimal thickness tumors because of the inefficiency of light in penetrating deep-seated tumors. To address this, the emerging field of radiation-activated PDT (radioPDT) uses X-rays to trigger photosensitizer-containing nanoparticles (NPs). A key consideration in radioPDT is the energy transfer efficiency from X-rays to the photosensitizer for ultimately generating the phototoxic reactive oxygen species (ROS). In this study, we developed a new variant of pegylated poly-lactic-co-glycolic (PEG-PLGA) encapsulated nanoscintillators (NSCs) along with a new, highly efficient ruthenium-based photosensitizer (Ru/radioPDT). Characterization of this NP via transmission electron microscopy, dynamic light scattering, UV-Vis spectroscopy, and inductively coupled plasma mass-spectroscopy showed an NP size of 120 nm, polydispersity index (PDI) of less than 0.25, high NSCs loading efficiency over 90% and in vitro accumulation within the cytosolic structure of endoplasmic reticulum and lysosome. The therapeutic efficacy of Ru/radioPDT was determined using PC3 cell viability and clonogenic assays. Ru/radioPDT exhibited minimal cell toxicity until activated by radiation to induce significant cancer cell kill over radiation alone. Compared to protoporphyrin IX-mediated radioPDT (PPIX/radioPDT), Ru/radioPDT showed higher capacity for singlet oxygen generation, maintaining a comparable cytotoxic effect on PC3 cells.

Using Biological Photophysics to Map the Excited-State Topology of Molecular Photosensitizers for Photodynamic Therapy.

This study employs TLD1433, a RuII -based photodynamic therapy (PDT) agent in human clinical trials, as a benchmark to establish protocols for studying the excited-state dynamics of photosensitizers (PSs) in cellulo, in the local environment provided by human cancer cells. Very little is known about the excited-state properties of any PS in live cells, and for TLD1433, it is terra incognita. This contribution targets a general problem in phototherapy, which is how to interrogate the light-triggered, function-determining processes of the PSs in the relevant biological environment, and establishes methodological advances to study the ultrafast photoinduced processes for TLD1433 when taken up by MCF7 cells. We generalize the methodological developments and results in terms of molecular physics by applying them to TLD1433's analogue TLD1633, making this study a benchmark to investigate the excited-state dynamics of phototoxic compounds in the complex biological environment.

Establishing a Robust and Reliable Response from a Potent Osmium-Based Photosensitizer Via Lipid Nanoformulation†.

Osmium (Os) based photosensitizers (PSs) are a unique class of nontetrapyrrolic metal-containing PSs that absorb red light. We recently reported a highly potent Os(II) PS, rac-[Os(phen)2 (IP-4T)](Cl)2 , referred to as ML18J03 herein, with light EC50 values as low as 20 pm. ML18J03 also exhibits low dark toxicity and submicromolar light EC50 values in hypoxia in some cell lines. However, owing to its longer oligothiophene chain, ML18J03 is not completely water soluble and forms 1-2 µm sized aggregates in PBS containing 1% DMSO. This aggregation causes variability in PDT efficacy between assays and thus unreliable and irreproducible reports of in vitro activity. To that end, we utilized PEG-modified DPPC liposomes (138 nm diameter) and DSPE-mPEG2000 micelles (10.2 nm diameter) as lipid nanoformulation vehicles to mitigate aggregation of ML18J03 and found that the spectroscopic properties important to biological activity were maintained or improved. Importantly, the lipid formulations decreased the interassay variance between the EC50 values by almost 20-fold, with respect to the unformulated ML18J03 when using broadband visible light excitation (P = 0.0276). Herein, lipid formulations are presented as reliable platforms for more accurate in vitro photocytotoxicity quantification for PSs prone to aggregation (such as ML18J03) and will be useful for assessing their in vivo PDT effects.

Enabling In Vivo Optical Imaging of an Osmium Photosensitizer by Micellar Formulation.

Osmium (Os)-based photosensitizers (PSs) exhibit unique broad, red-shifted absorption, favoring PDT activity at greater tissue depths. We recently reported on a potent Os(II) PS, rac-[Os(phen)2(IP-4T)](Cl)2 (ML18J03) with submicromolar hypoxia activity. ML18J03 exhibits a low luminescence quantum yield of 9.8 × 10-5 in PBS, which limits its capacity for in vivo luminescence imaging. We recently showed that formulating ML18J03 into 10.2 nm DSPE-mPEG2000 micelles (Mic-ML18J03) increases its luminescence quantum yield by two orders of magnitude. Here, we demonstrate that Mic-ML18J03 exhibits 47-fold improved accumulative luminescence signals in orthotopic AT-84 head and neck tumors. We show, for the first time, that micellar formulation provides up to 11.7-fold tumor selectivity for ML18J03. Furthermore, Mic-ML18J03 does not experience the concentration-dependent quenching observed with unformulated ML18J03 in PBS, and formulation reduces spectral shifting of the emission maxima during PDT (variance = 6.5 and 27.3, respectively). The Mic-ML18J03 formulation also increases the production of reactive molecular species 2-3-fold. These findings demonstrate that micellar formulation is a versatile and effective approach to enable in vivo luminescence imaging options for an otherwise quenched, yet promising, PS.

Insights into enantioselective separations of ionic metal complexes by sub/supercritical fluid chromatography.

Sub/supercritical fluid chromatography (SFC) is a green separation technique that has been used to separate a wide variety of compounds and is proven to be immensely useful for chiral separations. However, SFC is currently not thought to be applicable for ionic compounds due to their low solubility in CO2, even with additives and organic modifiers. Recently, a large amount of research has been centered on octahedral complexes of Ru(II) and Os(II) with bidentate polypyridyl ligands due to their ability to serve in cancer treatment and other biological activities. These compounds exist as the delta (Δ) and lambda (Λ) enantiomers. Previously, similar compounds have been enantiomerically separated using HPLC and capillary electrophoresis, but never with SFC. Cyclofructan-6 (CF6) derivatized with (R)-naphthyl ethyl (RN) groups has been proven to be an effective chiral stationary phase for these separations in HPLC. This column chemistry was expanded to SFC to provide the first chiral separation of a wide variety (23 complexes in total) of ionic octahedral polypyridyl complexes. Unexpected behavior for mixing methanol and acetonitrile as the organic modifier will be discussed, along with the effects of additives. Enantioselectivity on CF6-RN chemistry is shown to be dependent on the conjugation level and rigidity of the metal complexes. Mass transfer kinetic behavior is also shown, and high-efficiency baseline resolved rapid separations are shown for fast screening or quantitation of representative coordination complexes.

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.

Ru(II) CONTAINING PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY: A CRITIQUE ON REPORTING AND AN ATTEMPT TO COMPARE EFFICACY.

Ruthenium(II)-based coordination complexes have emerged as photosensitizers (PSs) for photodynamic therapy (PDT) in oncology as well as antimicrobial indications and have great potential. Their modular architectures that integrate multiple ligands can be exploited to tune cellular uptake and subcellular targeting, solubility, light absorption, and other photophysical properties. A wide range of Ru(II) containing compounds have been reported as PSs for PDT or as photochemotherapy (PCT) agents. Many studies employ a common scaffold that is subject to systematic variation in one or two ligands to elucidate the impact of these modifications on the photophysical and photobiological performance. Studies that probe the excited state energies and dynamics within these molecules are of fundamental interest and are used to design next-generation systems. However, a comparison of the PDT efficacy between Ru(II) containing PSs and 1st or 2nd generation PSs, already in clinical use or preclinical/clinical studies, is rare. Even comparisons between Ru(II) containing molecular structures are difficult, given the wide range of excitation wavelengths, power densities, and cell lines utilized. Despite this gap, PDT dose metrics quantifying a PS's efficacy are available to perform qualitative comparisons. Such models are independent of excitation wavelength and are based on common outcome parameters, such as the photon density absorbed by the Ru(II) compound to cause 50% cell kill (LD50) based on the previously established threshold model. In this focused photophysical review, we identified all published studies on Ru(II) containing PSs since 2005 that reported the required photophysical, light treatment, and in vitro outcome data to permit the application of the Photodynamic Threshold Model to quantify their potential efficacy. The resulting LD50 values range from less than 1013 to above 1020 [hν cm-3], indicating a wide range in PDT efficacy and required optical energy density for ultimate clinical translation.