Photodynamic Inactivation of Human Coronaviruses

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses

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

Synthesis and Photobiological Activity of Ru(II) Dyads Derived from Pyrrole-2-carboxylate Thionoesters

The synthesis and characterization of a series of heteroleptic ruthenium­(II) dyads derived from pyrrole-2-carboxylate thionoesters are reported. Ligands bearing a conjugated thiocarbonyl group were found to be more reactive toward Ru­(II) complexation compared to analogous all-oxygen pyrrole-2-carboxylate esters, and salient features of the resulting complexes were determined using X-ray crystallography, electronic absorption, and NMR spectroscopy. Selected complexes were evaluated for their potential in photobiological applications, whereupon all compounds demonstrated in vitro photodynamic therapy effects in HL-60 and SK-MEL-28 cells, with low nanomolar activities observed, and exhibited some of the largest photocytotoxicity indices to date (>2000). Importantly, the Ru­(II) dyads could be activated by relatively soft doses of visible (100 J cm^–2, 29 mW cm^–2) or red light (100 J cm^–2, 34 mW cm^–2), which is compatible with therapeutic applications. Some compounds even demonstrated up to five-fold selectivity for malignant cells over noncancerous cells. These complexes were also shown to photocleave, and in some cases unwind, DNA in cell-free experiments. Thus, this new class of Ru­(II) dyads has the capacity to interact with and damage biological macromolecules in the cell, making them attractive agents for photodynamic therapy

Photodynamic Inactivation of Herpes Simplex Viruses

Herpes simplex virus (HSV) infections can be treated with direct acting antivirals like acyclovir and foscarnet, but long-term use can lead to drug resistance, which motivates research into broadly-acting antivirals that can provide a greater genetic barrier to resistance. Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species that inactivate microorganisms. The botanical plant extract OrthoquinTM is a powerful photosensitizer with antimicrobial properties. Here we report that Orthoquin also has antiviral properties. Photoactivated Orthoquin inhibited herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) infection of target cells in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. HSV inactivation required direct contact between Orthoquin and the inoculum, whereas pre-treatment of target cells had no effect. Orthoquin did not cause appreciable damage to viral capsids or premature release of viral genomes, as measured by qPCR for the HSV-1 genome. By contrast, immunoblotting for HSV-1 antigens in purified virion preparations suggested that higher doses of Orthoquin had a physical impact on certain HSV-1 proteins that altered protein mobility or antigen detection. Orthoquin PDI also inhibited the non-enveloped adenovirus (AdV) in a dose-dependent manner, whereas Orthoquin-mediated inhibition of the enveloped vesicular stomatitis virus (VSV) was light-independent. Together, these findings suggest that the broad antiviral effects of Orthoquin-mediated PDI may stem from damage to viral attachment proteins

π‑Expansive Heteroleptic Ruthenium(II) Complexes as Reverse Saturable Absorbers and Photosensitizers for Photodynamic Therapy

Five heteroleptic tris-diimine ruthenium­(II) complexes [RuL­(N^N)₂]­(PF₆)₂ (where <b>L</b> is 3,8-di­(benzothiazolylfluorenyl)-1,10-phenanthroline and N^N is 2,2′-bipyridine (bpy) (<b>1</b>), 1,10-phenanthroline (phen) (<b>2</b>), 1,4,8,9-tetraazatriphenylene (tatp) (<b>3</b>), dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine (dppz) (<b>4</b>), or benzo­[<i>i</i>]­dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine (dppn) (<b>5</b>), respectively) were synthesized. The influence of π-conjugation of the ancillary ligands (N^N) on the photophysical properties of the complexes was investigated by spectroscopic methods and simulated by density functional theory (DFT) and time-dependent DFT. Their ground-state absorption spectra were characterized by intense absorption bands below 350 nm (ligand <b>L</b> localized ¹<i>π,π</i>* transitions) and a featureless band centered at ∼410 nm (intraligand charge transfer (¹ILCT)/¹<i>π,π</i>* transitions with minor contribution from metal-to-ligand charge transfer (¹MLCT) transition). For complexes <b>4</b> and <b>5</b> with dppz and dppn ligands, respectively, broad but very weak absorption (ε < 800 M^–1 cm^–1) was present from 600 to 850 nm, likely emanating from the spin-forbidden transitions to the triplet excited states. All five complexes showed red-orange phosphorescence at room temperature in CH₂Cl₂ solution with decreased lifetimes and emission quantum yields, as the π-conjugation of the ancillary ligands increased. Transient absorption (TA) profiles were probed in acetonitrile solutions at room temperature for all of the complexes. Except for complex <b>5</b> (which showed dppn-localized ³<i>π,π</i>* absorption with a long lifetime of 41.2 μs), complexes <b>1</b>–<b>4</b> displayed similar TA spectral features but with much shorter triplet lifetimes (1–2 μs). Reverse saturable absorption (RSA) was demonstrated for the complexes at 532 nm using 4.1 ns laser pulses, and the strength of RSA decreased in the order: <b>2</b> ≥ <b>1</b> ≈ <b>5</b> > <b>3</b> > <b>4</b>. Complex <b>5</b> is particularly attractive as a broadband reverse saturable absorber due to its wide optical window (430–850 nm) and long-lived triplet lifetime in addition to its strong RSA at 532 nm. Complexes <b>1</b>–<b>5</b> were also probed as photosensitizing agents for in vitro photodynamic therapy (PDT). Most of them showed a PDT effect, and <b>5</b> emerged as the most potent complex with red light (EC₅₀ = 10 μM) and was highly photoselective for melanoma cells (selectivity factor, SF = 13). Complexes <b>1</b>–<b>5</b> were readily taken up by cells and tracked by their intracellular luminescence before and after a light treatment. Diagnostic intracellular luminescence increased with increased π-conjugation of the ancillary N^N ligands despite diminishing cell-free phosphorescence in that order. All of the complexes penetrated the nucleus and caused DNA condensation in cell-free conditions in a concentration-dependent manner, which was not influenced by the identity of N^N ligands. Although the mechanism for photobiological activity was not established, complexes <b>1</b>–<b>5</b> were shown to exhibit potential as theranostic agents. Together the RSA and PDT studies indicate that developing new agents with long intrinsic triplet lifetimes, high yields for triplet formation, and broad ground-state absorption to near-infrared (NIR) in tandem is a viable approach to identifying promising agents for these 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