Excited State Dynamics of a Photobiologically Active Ru(II) Dyad Are Altered in Biologically Relevant Environments

In this study femtosecond and nanosecond time-resolved transient absorption spectroscopy was used to investigate the influence of ionic strength and complexity on the excited state dynamics of a Ru­(II)-based metal–organic dyad. The bis-heteroleptic complex [Ru­(bpy)₂(ippy)]²⁺ (<b>1</b>), where bpy = 2,2′-bipyridine and ippy = 2-(1-pyrenyl-1<i>H-</i>imidazo­[4,5-<i>f</i>]­[1,10]­phenanthroline, is a potent photosensitizer for in vitro photodynamic therapy (PDT) and photodynamic inactivation (PDI) of microorganisms owing to a long-lived triplet excited state derived from a metal-to-ligand charge-transfer (³MLCT) state that is equilibrium with an intraligand (³IL) state. The prolonged lifetime provides ample opportunity for bimolecular quenching of this state by oxygen; thus singlet oxygen (¹O₂) sensitization is very efficient. In simple aqueous solution, fast cooling within the ³MLCT manifold is followed by energy transfer to an ³IL state, which is facilitated by rotation of a pyrenyl unit about the imidazo–pyrenyl (ip) coannular bond. For solutions of <b>1</b> in high ionic strength simulated biological fluid (SBF), a more physiologically relevant solvent that contains a complex mixture of ions at pH 7.4, femtosecond studies revealed an additional excited state, possibly based on an ion–ligand interaction. This new state appearing in high ionic strength SBF was not observable in water, simple buffers, or low ionic strength SBF. These photoinduced dynamics were also affected by the presence of biomolecules such as DNA in simple buffer, whereby relaxation on the picosecond time scale was accelerated from 39 to 18 ps with DNA intercalation by <b>1</b>. The increased rate of coplanarization of the pyrene and the imidazole units was attributed to DNA-induced conformational restriction of the pyrenyl unit relative to the ip bond. Quantitative changes to excited state decay rates of <b>1</b> in solutions of high ionic strength were also observed when probed on the microsecond time scale. Notably, the thermalized excited state decay pathways were altered substantially with DNA intercalation, with access to some states being completely blocked. Experimentally, this manifested in the absence of the slowest microsecond decay channel, which is normally observed for <b>1</b> in solution. The quantitative and qualitative observations from this study highlight the importance of employing biologically relevant solvents and potential biomolecule targets when the excited state dynamics and photophysical properties (under cell-free conditions) responsible for the potent photobiological effects are assessed in the context of photodynamic therapy and photodynamic inactivation

Organometallic Ru(II) Photosensitizers Derived from π‑Expansive Cyclometalating Ligands: Surprising Theranostic PDT Effects

The purpose of the present study was to investigate the influence of π-expansive cyclometalating ligands on the photophysical and photobiological properties of organometallic Ru­(II) compounds. Four compounds with increasing π conjugation on the cyclometalating ligand were prepared, and their structures were confirmed by HPLC, 1D and 2D ¹H NMR, and mass spectrometry. The properties of these compounds differed substantially from their Ru­(II) polypyridyl counterparts. Namely, they were characterized by red-shifted absorption, very weak to no room temperature phosphorescence, extremely short phosphorescence state lifetimes (<10 ns), low singlet oxygen quantum yields (0.5–8%), and efficient ligand-centered fluorescence. Three of the metal complexes were very cytotoxic to cancer cells in the dark (EC₅₀ values = 1–2 μM), in agreement with what has traditionally been observed for Ru­(II) compounds derived from small C^N ligands. Surprisingly, the complex derived from the most π-expansive cyclometalating ligand exhibited no cytotoxicity in the dark (EC₅₀ > 300 μM) but was phototoxic to cells in the nanomolar regime. Exceptionally large phototherapeutic margins, exceeding 3 orders of magnitude in some cases, were accompanied by bright ligand-centered intracellular fluorescence in cancer cells. Thus, Ru­(II) organometallic systems derived from π-expansive cyclometalating ligands, such 4,9,16-triazadibenzo­[<i>a,c</i>]­napthacene (pbpn), represent the first class of potent light-responsive Ru­(II) cyclometalating agents with theranostic potential

<i>In Vitro</i> Multiwavelength PDT with ³IL States: Teaching Old Molecules New Tricks

The purpose of the present investigation was to ascertain whether ³IL excited states with microsecond lifetimes are universally potent for photodynamic applications, and if these long-lived states are superior to their ³MLCT counterparts as <i>in vitro</i> PDT agents. A family of blue-green absorbing, Ru­(II)-based transition metal complexes derived from the π-expansive dppn ligand was prepared and characterized according to its photodynamic activity against HL-60 cells, and toward DNA in cell-free media. Complexes in this series that are characterized by low-energy and long-lived ³IL excited states photocleaved DNA with blue, green, red, and near-IR light. This panchromatic photodynamic effect translated to <i>in vitro</i> multiwavelength photodynamic therapy (PDT) with red-light cytotoxicities as low as 1.5 μM (EC₅₀) for the parent complex and 400 nM for its more lipophilic counterpart. This potency is similar to that achieved with Ru­(II)-based dyads containing long-lived ³IL excitons located on appended pyrenyl units, and appears to be a general property of sufficiently long-lived excited states. Moreover, the red PDT observed for certain members of this family was almost 5 times more potent than Photofrin with therapeutic indices 30 times greater. Related Ru­(II) complexes having lowest-lying ³MLCT states of much shorter duration (≤1 μs) did not yield DNA photodamage or <i>in vitro</i> PDT with red or near-IR light, nor did the corresponding Os­(II) complex with a submicrosecond ³IL excited state lifetime. Therefore, metal complexes that utilize highly photosensitizing ³IL excited states, with suitably long lifetimes (≫ 1 μs), are well-poised to elicit PDT at wavelengths even where their molar extinction coefficients are very low (<100 M^–1 cm^–1). Herein we demonstrate that such unexpected reactivity gives rise to very effective PDT in the typical therapeutic window (600–850 nm)

Predictive Strength of Photophysical Measurements for in Vitro Photobiological Activity in a Series of Ru(II) Polypyridyl Complexes Derived from π-Extended Ligands

This study investigates the correlation between photocytotoxicity and the prolonged excited-state lifetimes exhibited by certain Ru­(II) polypyridyl photosensitizers comprised of π-expansive ligands. The eight metal complexes selected for this study differ markedly in their triplet state configurations and lifetimes. Human melanoma SKMEL28 and human leukemia HL60 cells were used as in vitro models to test photocytotoxicity induced by the compounds when activated by either broadband visible or monochromatic red light. The photocytotoxicities of the metal complexes investigated varied over 2 orders of magnitude and were positively correlated with their excited-state lifetimes. The complexes with the longest excited-state lifetimes, contributed by low-lying 3IL states, were the most phototoxic toward cancer cells under all conditions

Photophysics of Ru(II) Dyads Derived from Pyrenyl-Substitued Imidazo[4,5‑<i>f</i>][1,10]phenanthroline Ligands

The photophysics of a series of Ru­(II) dyads based on the 2-(1-pyrenyl)-1<i>H</i>-imidazo­[4,5-<i>f</i>]­[1,10]-phenanthroline ligand was investigated. The ability of these metal complexes to intercalate DNA and induce cell death upon photoactivation makes them attractive photosensitizers for a range of photobiological applications, including photodynamic therapy. In the present study, time-resolved transient absorption and emission spectroscopy were used to interrogate the photoinduced processes that follow metal-to-ligand charge transfer excitation of the complexes in solution. It was found that energy transfer to pyrene-localized intraligand triplet states, facilitated by torsional motion of the pyrene moiety relative to the imidazo­[4,5-<i>f</i>]­[1,10]­phenanthroline ligand, was an important relaxation pathway governing the photophysical dynamics in this class of compounds. Biphasic decay kinetics were assigned to spontaneous (pre-equilibrium) and delayed emission, arising from an equilibrium established between ³MLCT and ³IL states. TDDFT calculations supported these interpretations

Influence of Protonation State on the Excited State Dynamics of a Photobiologically Active Ru(II) Dyad

The influence of ligand protonation on the photophysics of a ruthenium (Ru) dyad bearing the 2-(1-pyrenyl)-1<i>H</i>-imidazo­[4,5-<i>f</i>]­[1,10]-phenanthroline (ippy) ligand was investigated by time-resolved transient absorption spectroscopy. It was found that changes in the protonation state of the imidazole group led to changes in the electronic configuration of the lowest lying excited state. Formation of the fully deprotonated imidazole anion resulted in excited state signatures that were consistent with a low-lying intraligand (IL) triplet state. This assignment was supported by time-dependent density functional theory (TDDFT) calculations. IL triplet states have been suggested to be potent mediators of photodynamic effects. Thus, these results are of interest in the design of Ru metal complexes as photosensitizers (PSs) for photodynamic therapy (PDT)

Ru(II) Dyads Derived from 2‑(1-Pyrenyl)‑1<i>H</i>‑imidazo[4,5‑<i>f</i>][1,10]phenanthroline: Versatile Photosensitizers for Photodynamic Applications

Combining the best attributes of organic photosensitizers with those of coordination complexes is an elegant way to achieve prolonged excited state lifetimes in Ru­(II) dyads. Not only do their reduced radiative and nonradiative rates provide ample time for photosensitization of reactive oxygen species at low oxygen tension but they also harness the unique properties of ³IL states that can act as discrete units or in concert with ³MLCT states. The imidazo­[4,5-<i>f</i>]­[1,10]­phenanthroline framework provides a convenient tether for linking π-expansive ligands such as pyrene to a Ru­(II) scaffold, and the stabilizing coligands can fine-tune the chemical and biological properties of these bichromophoric systems. The resulting dyads described in this study exhibited nanomolar light cytotoxicities against cancer cells with photocytotoxicity indices exceeding 400 for some coligands employed. This potency extended to bacteria, where concentrations as low as 10 nM destroyed 75% of a bacterial population. Notably, these dyads remained extremely active against biofilm with light photocytotoxicities against these more resistant bacterial populations in the 10–100 nM regime. The results from this study demonstrate the versatility of these highly potent photosensitizers in destroying both cancer and bacterial cells and expand the scope of compounds that utilize low-lying ³IL states for photobiological applications

Cyclometalated Ruthenium(II) Complexes Derived from α‑Oligothiophenes as Highly Selective Cytotoxic or Photocytotoxic Agents

The photophysical and photobiological properties of a new class of cyclometalated ruthenium­(II) compounds incorporating π-extended benzo­[<i>h</i>]­imidazo­[4,5-<i>f</i>]­quinoline (IBQ) cyclometalating ligands (C^N) bearing thienyl rings (<i>n</i> = 1–4, compounds <b>1</b>–<b>4</b>) were investigated. Their octanol–water partition coefficients (log <i>P</i>_o/w) were positive and increased with <i>n</i>. Their absorption and emission energies were red-shifted substantially compared to the analogous Ru­(II) diimine (N^N) complexes. They displayed C^N-based intraligand (IL) fluorescence and triplet excited-state absorption that shifted to longer wavelengths with increasing <i>n</i> and N^N-based metal-to-ligand charge transfer (MLCT) phosphorescence that was independent of <i>n</i>. Their photoluminescence lifetimes (τ_em) ranged from 4–10 ns for ¹IL states and 12–18 ns for ³MLCT states. Transient absorption lifetimes (τ_TA) were 5–8 μs with 355 nm excitation, ascribed to ³IL states that became inaccessible for <b>1</b>–<b>3</b> with 532 nm excitation (<b>1</b>–<b>3</b>, τ_TA = 16–17 ns); the ³IL of <b>4</b> only was accessible by lower energy excitation, τ_TA = 3.8 μs. Complex <b>4</b> was nontoxic (EC₅₀ > 300 μM) to SK-MEL-28 melanoma cells and CCD1064-Sk normal skin fibroblasts in the dark, while <b>3</b> was selectively cytotoxic to melanoma (EC₅₀= 5.1 μM) only. Compounds <b>1</b> and <b>2</b> were selective for melanoma cells in the dark, with submicromolar potencies (EC₅₀ = 350–500 nM) and selectivity factors (SFs) around 50. The photocytotoxicities of compounds <b>1</b>–<b>4</b> toward melanoma cells were similar, but only compounds <b>3</b> and <b>4</b> displayed significant phototherapeutic indices (PIs; <b>3</b>, 43; <b>4</b>, >1100). The larger cytotoxicities for compounds <b>1</b> and <b>2</b> were attributed to increased cellular uptake and nuclear accumulation, and possibly related to the DNA-aggregating properties of all four compounds as demonstrated by cell-free gel mobility-shift assays. Together, these results demonstrate a new class of thiophene-containing Ru­(II) cyclometalated compounds that contain both highly selective chemotherapeutic agents and extremely potent photocytotoxic agents