Ligand Rigidity Steers the Selectivity and Efficiency of the Photosubstitution Reaction of Strained Ruthenium Polypyridyl Complexes.

While photosubstitution reactions in metal complexes are usually thought of as dissociative processes poorly dependent on the environment, they are, in fact, very sensitive to solvent effects. Therefore, it is crucial to explicitly consider solvent molecules in theoretical models of these reactions. Here, we experimentally and computationally investigated the selectivity of the photosubstitution of diimine chelates in a series of sterically strained ruthenium(II) polypyridyl complexes in water and acetonitrile. The complexes differ essentially by the rigidity of the chelates, which strongly influenced the observed selectivity of the photosubstitution. As the ratio between the different photoproducts was also influenced by the solvent, we developed a full density functional theory modeling of the reaction mechanism that included explicit solvent molecules. Three reaction pathways leading to photodissociation were identified on the triplet hypersurface, each characterized by either one or two energy barriers. Photodissociation in water was promoted by a proton transfer in the triplet state, which was facilitated by the dissociated pyridine ring acting as a pendent base. We show that the temperature variation of the photosubstitution quantum yield is an excellent tool to compare theory with experiments. An unusual phenomenon was observed for one of the compounds in acetonitrile, for which an increase in temperature led to a surprising decrease in the photosubstitution reaction rate. We interpret this experimental observation based on complete mapping of the triplet hypersurface of this complex, revealing thermal deactivation to the singlet ground state through intersystem crossing.

Understanding and Preventing Photoluminescence Quenching to Achieve Unity Photoluminescence Quantum Yield in Yb:YLF Nanocrystals.

Ytterbium-doped LiYF4 (Yb:YLF) is a commonly used material for laser applications, as a photon upconversion medium, and for optical refrigeration. As nanocrystals (NCs), the material is also of interest for biological and physical applications. Unfortunately, as with most phosphors, with the reduction in size comes a large reduction of the photoluminescence quantum yield (PLQY), which is typically associated with an increase in surface-related PL quenching. Here, we report the synthesis of bipyramidal Yb:YLF NCs with a short axis of ∼60 nm. We systematically study and remove all sources of PL quenching in these NCs. By chemically removing all traces of water from the reaction mixture, we obtain NCs that exhibit a near-unity PLQY for an Yb3+ concentration below 20%. At higher Yb3+ concentrations, efficient concentration quenching occurs. The surface PL quenching is mitigated by growing an undoped YLF shell around the NC core, resulting in near-unity PLQY values even for fully Yb3+-based LiYbF4 cores. This unambiguously shows that the only remaining quenching sites in core-only Yb:YLF NCs reside on the surface and that concentration quenching is due to energy transfer to the surface. Monte Carlo simulations can reproduce the concentration dependence of the PLQY. Surprisingly, Förster resonance energy transfer does not give satisfactory agreement with the experimental data, whereas nearest-neighbor energy transfer does. This work demonstrates that Yb3+-based nanophosphors can be synthesized with a quality close to that of bulk single crystals. The high Yb3+ concentration in the LiYbF4/LiYF4 core/shell nanocrystals increases the weak Yb3+ absorption, making these materials highly promising for fundamental studies and increasing their effectiveness in bioapplications and optical refrigeration.

Xanthoepocin, a photolabile antibiotic of Penicillium ochrochloron CBS 123823 with high activity against multiresistant gram-positive bacteria.

BACKGROUND: With the steady increase of antibiotic resistance, several strategies have been proposed in the scientific community to overcome the crisis. One of many successful strategies is the re-evaluation of known compounds, which have been early discarded out of the pipeline, with state-of-the-art know-how. Xanthoepocin, a polyketide widespread among the genus Penicillium with an interesting bioactivity spectrum against gram-positive bacteria, is such a discarded antibiotic. The purpose of this work was to (i) isolate larger quantities of this metabolite and chemically re-evaluate it with modern technology, (ii) to explore which factors lead to xanthoepocin biosynthesis in P. ochrochloron, and (iii) to test if it is beside its known activity against methicillin-resistant Staphylococcus aureus (MRSA), also active against linezolid and vancomycin-resistant Enterococcus faecium (LVRE)-a very problematic resistant bacterium which is currently on the rise. RESULTS: In this work, we developed several new protocols to isolate, extract, and quantify xanthoepocin out of bioreactor batch and petri dish-grown mycelium of P. ochrochloron. The (photo)chemical re-evaluation with state-of-the-art techniques revealed that xanthoepocin is a photolabile molecule, which produces singlet oxygen under blue light irradiation. The intracellular xanthoepocin content, which was highest under ammonium-limited conditions, varied considerably with the applied irradiation conditions in petri dish and bioreactor batch cultures. Using light-protecting measures, we achieved MIC values against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which were up to 5 times lower than previously published. In addition, xanthoepocin was highly active against a clinical isolate of linezolid and vancomycin-resistant Enterococcus faecium (LVRE). CONCLUSIONS: This interdisciplinary work underlines that the re-evaluation of known compounds with state-of-the-art techniques is an important strategy in the combat against multiresistant bacteria and that light is a crucial factor on many levels that needs to receive more attention. With appropriate light protecting measures in the susceptibility tests, xanthoepocin proved to be a powerful antibiotic against MRSA and LVRE. Exploring the light response of other polyketides may be pivotal for re-introducing previously discarded metabolites into the antibiotic pipeline and to identify photosensitizers which might be used for (antimicrobial) photodynamic therapies.

Light-triggered switching of liposome surface charge directs delivery of membrane impermeable payloads in vivo.

Surface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching of liposome surface charge, from neutral to cationic, in situ and in vivo (embryonic zebrafish). Prior to light activation, intravenously administered liposomes, composed of just two lipid reagents, freely circulate and successfully evade innate immune cells present in the fish. Upon in situ irradiation and surface charge switching, however, liposomes rapidly adsorb to, and are taken up by, endothelial cells and/or are phagocytosed by blood resident macrophages. Coupling complete external control of nanoparticle targeting together with the intracellular delivery of encapsulated (and membrane impermeable) cargos, these compositionally simple liposomes are proof that advanced nanoparticle function in vivo does not require increased design complexity but rather a thorough understanding of the fundamental nano-bio interactions involved.

796 nm Activation of a Photocleavable Ruthenium(II) Complex Conjugated to an Upconverting Nanoparticle through Two Phosphonate Groups.

The biological application of photoactivatable ruthenium anticancer prodrugs is limited by the need to use poorly penetrating high-energy visible light for their activation. Upconverting nanoparticles (UCNPs), which produce high-energy light under near-infrared (NIR) excitation, can solve this issue, provided that they form stable, water (H2O)-dispersible nanoconjugates with the prodrug and that there is efficient energy transfer from the UCNP to the ruthenium complex. Herein, we report on the synthesis and photochemistry of the ruthenium(II) polypyridyl complex [Ru(bpy)2(3H)](PF6)2 ([1](PF6)2), where bpy = 2,2-bipyridine and 3H is a photocleavable bis(thioether) ligand modified with two phosphonate moieties. This ligand was coordinated to the ruthenium center through its thioether groups and could be dissociated under blue-light irradiation. Complex [1](PF6)2 was bound to the surface of NaYF4:Yb3+,Tm3+@NaYF4:Nd3+@NaYF4 core-shell-shell (CSS-)UCNPs through its bis(phosphonate) group, thereby creating a H2O-dispersible, thermally stable nanoconjugate (CSS-UCNP@[1]). Conjugation to the nanoparticle surface was found to be most efficient in neutral to slightly basic conditions, resulting in up to 2.4 × 103 RuII ions per UCNP. The incorporation of a neodymium-doped shell layer allowed for the generation of blue light using low-energy, deep-penetrating light (796 nm). This wavelength prevents the undesired heating seen with conventional UCNPs activated at 980 nm. Irradiation of CSS-UCNP@[1] with NIR light led to activation of the ruthenium complex [1](PF6)2. Although only one of the two thioether groups was dissociated under irradiation at 50 W·cm-2, we provide the first demonstration of the photoactivation of a ruthenium thioether complex using 796 nm irradiation of a H2O-dispersible nanoconjugate.

Diastereoselective Synthesis and Two-Step Photocleavage of Ruthenium Polypyridyl Complexes Bearing a Bis(thioether) Ligand.

Thioethers are good ligands for photoactivatable ruthenium(II) polypyridyl complexes, as they form thermally stable complexes that are prone to ligand photosubstitution. Here, we introduce a novel symmetric chelating bis(thioether) ligand scaffold, based on 1,3-bis(methylthio)-2-propanol (4) and report the synthesis and stereochemical characterization of the series of novel ruthenium(II) polypyridyl complexes [Ru(bpy)2(L)](PF6)2 ([1]-[3](PF6)2), where L is ligand 4, its methyl ether, 1,3-bis(methylthio)-2-methoxypropane (5), or its carboxymethyl ether, 1,3-bis(methylthio)-2-(carboxymethoxy)propane (6). Coordination of ligands 4-6 to the bis(bipyridine)ruthenium center gives rise to 16 possible isomers, consisting of 8 possible Λ diastereoisomers and their Δ enantiomers. We found that the synthesis of [1]-[3](PF6)2 is diastereoselective, yielding a racemic mixture of the Λ-(S)-eq-(S)-ax-OHeq-[Ru]2+ and Δ-(R)-ax-(R)-eq-OHeq-[Ru]2+ isomers. Upon irradiation with blue light in water, [1]-[3](PF6)2 selectively substitute their bis(thioether) ligands for water molecules in a two-step photoreaction, ultimately producing [Ru(bpy)2(H2O)2]2+ as the photoproduct. The relatively stable photochemical intermediate was identified as cis-[Ru(bpy)2(κ1-L)(H2O)]2+ by mass spectrometry. Global fitting of the time evolution of the UV-vis absorption spectra of [1]-[3](PF6)2 was employed to derive the photosubstitution quantum yields (Φ443) for each of the two photochemical reaction steps separately, revealing very high quantum yields of 0.16-0.25 for the first step and lower values (0.0055-0.0093) for the second step of the photoreaction. The selective and efficient photochemical reaction makes the photocleavable bis(thioether) ligand scaffold reported here a promising candidate for use in e.g. ruthenium-based photo-activated chemotherapy.

NIR-Light-Driven Generation of Reactive Oxygen Species Using Ru(II)-Decorated Lipid-Encapsulated Upconverting Nanoparticles.

The biological application of ruthenium anticancer prodrugs for photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) is restricted by the need to use poorly penetrating high-energy photons for their activation, i.e., typically blue or green light. Upconverting nanoparticles (UCNPs), which produce high-energy light under near-infrared (NIR) excitation, may solve this issue, provided that the coupling between the UCNP surface and the Ru prodrug is optimized to produce stable nanoconjugates with efficient energy transfer from the UCNP to the ruthenium complex. Herein, we report on the synthesis and photochemistry of the two structurally related ruthenium(II) polypyridyl complexes [Ru(bpy)2(5)](PF6)2 ([1](PF6)2) and [Ru(bpy)2(6)](PF6)2 ([2](PF6)2), where bpy = 2,2-bipyridine, 5 is 5,6-bis(dodecyloxy)-2,9-dimethyl-1,10-phenanthroline, and 6 is 5,6-bis(dodecyloxy)-1,10-phenanthroline. [1](PF6)2 is photolabile as a result of the steric strain induced by ligand 5, but the irradiation of [1](PF6)2 in solution leads to the nonselective and slow photosubstitution of one of its three ligands, making it a poor PACT compound. On the other hand, [2](PF6)2 is an efficient and photostable PDT photosensitizer. The water-dispersible, negatively charged nanoconjugate UCNP@lipid/[2] was prepared by the encapsulation of 44 nm diameter NaYF4:Yb3+,Tm3+ UCNPs in a mixture of 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycero-3-phosphocholine phospholipids, cholesterol, and the amphiphilic complex [2](PF6)2. A nonradiative energy transfer efficiency of 12% between the Tm3+ ions in the UCNP and the Ru2+ acceptor [2]2+ was found using time-resolved emission spectroscopy. Under irradiation with NIR light (969 nm), UCNP@lipid/[2] was found to produce reactive oxygen species (ROS), as judged by the oxidation of the nonspecific ROS probe 2',7'-dichlorodihydrofluorescein (DCFH2-). Determination of the type of ROS produced was precluded by the negative surface charge of the nanoconjugate, which resulted in the electrostatic repulsion of the more specific but also negatively charged 1O2 probe tetrasodium 9,10-anthracenediyl-bis(methylene)dimalonate (Na4(ADMBMA)).

The two isomers of a cyclometallated palladium sensitizer show different photodynamic properties in cancer cells.

This report demonstrates that changing the position of the carbon-metal bond in a polypyridyl cyclopalladated complex, i.e. going from PdL1 (N^N^C^N) to PdL2 (N^N^N^C), dramatically influences the photodynamic properties of the complex in cancer cells. This effect is primarily attributed to the significantly difference in absorbance and singlet oxygen quantum yields between the two isomers.

Absolute upconversion quantum yields of blue-emitting LiYF4:Yb3+,Tm3+ upconverting nanoparticles.

The upconversion quantum yield (ΦUC) is an essential parameter for the characterization of the optical performance of lanthanoid-doped upconverting nanoparticles (UCNPs). Despite its nonlinear dependence on excitation power density (Pexc), it is typically reported only as a single number. Here, we present the first measurement of absolute upconversion quantum yields of the individual emission bands of blue light-emitting LiYF4:Yb3+,Tm3+ UCNPs in toluene. Reporting the quantum yields for the individual emission bands is required for assessing the usability of UCNPs in various applications that require upconverted light of different wavelengths, such as bioimaging, photocatalysis and phototherapy. Here, the reliability of the ΦUC measurements is demonstrated by studying the same batch of UCNPs in three different research groups. The results show that whereas the total upconversion quantum yield of these UCNPs is quite high-typically 0.02 at a power density of 5 W cm-2-most of the upconverted photon flux is emitted in the 794 nm upconversion band, while the blue emission band at 480 nm is very weak, with a much lower quantum yield of ∼6 × 10-5 at 5 W cm-2. Overall, although the total upconversion quantum yield of LiYF4:Yb3+,Tm3+ UCNPs seems satisfying, notably for NIR bioimaging, blue-light demanding phototherapy applications will require better-performing UCNPs with higher blue light upconversion quantum yields.

Dynamics of dual-fluorescent polymersomes with durable integrity in living cancer cells and zebrafish embryos.

The long-term fate of biomedical nanoparticles after endocytosis is often only sparsely addressed in vitro and in vivo, while this is a crucial parameter to conclude on their utility. In this study, dual-fluorescent polyisobutylene-polyethylene glycol (PiB-PEG) polymersomes were studied for several days in vitro and in vivo. In order to optically track the vesicles' integrity, one fluorescent probe was located in the membrane and the other in the aqueous interior compartment. These non-toxic nanovesicles were quickly endocytosed in living A549 lung carcinoma cells but unusually slowly transported to perinuclear lysosomal compartments, where they remained intact and luminescent for at least 90 h without being exocytosed. Fluorescence-assisted flow cytometry indicated that after endocytosis, the nanovesicles were eventually degraded within 7-11 days. In zebrafish embryos, the polymersomes caused no lethality and were quickly taken up by the endothelial cells, where they remained fully intact for as long as 96 h post-injection. This work represents a novel case-study of the remarkable potential of PiB-PEG polymersomes as an in vivo bio-imaging and slow drug delivery platform.

A Red-Light-Activated Ruthenium-Caged NAMPT Inhibitor Remains Phototoxic in Hypoxic Cancer Cells.

We describe two water-soluble ruthenium complexes, [1]Cl2 and [2]Cl2 , that photodissociate to release a cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with a low dose (21 J cm-2 ) of red light in an oxygen-independent manner. Using a specific NAMPT activity assay, up to an 18-fold increase in inhibition potency was measured upon red-light activation of [2]Cl2 , while [1]Cl2 was thermally unstable. For the first time, the dark and red-light-induced cytotoxicity of these photocaged compounds could be tested under hypoxia (1 % O2 ). In skin (A431) and lung (A549) cancer cells, a 3- to 4-fold increase in cytotoxicity was found upon red-light irradiation for [2]Cl2 , whether the cells were cultured and irradiated with 1 % or 21 % O2 . These results demonstrate the potential of photoactivated chemotherapy for hypoxic cancer cells, in which classical photodynamic therapy, which relies on oxygen activation, is poorly efficient.

To cage or to be caged? The cytotoxic species in ruthenium-based photoactivated chemotherapy is not always the metal.

In metal-based photoactivated chemotherapy (PACT), two photoproducts are generated by light-triggered photosubstitution of a metal-bound ligand: the free ligand itself and an aquated metal complex. By analogy with cisplatin, the aquated metal complex is usually presented as the biologically active species, as it can typically bind to DNA. In this work, we show that this qualitative assumption is not necessarily valid by comparing the biological activity, log P, and cellular uptake of three ruthenium-based PACT complexes: [Ru(bpy)2(dmbpy)]2+, [Ru(bpy)2(mtmp)]2+, and [Ru(Ph2phen)2(mtmp)]2+. For the first complex, the photoreleased dmbpy ligand is responsible for the observed phototoxicity, whereas the second complex is not phototoxic, and for the third complex it is the ruthenium bis-aqua photoproduct that is the sole cytotoxic species.

Red Light Activation of Ru(II) Polypyridyl Prodrugs via Triplet-Triplet Annihilation Upconversion: Feasibility in Air and through Meat.

Triplet-triplet annihilation upconversion (TTA-UC) is a promising photophysical tool to shift the activation wavelength of photopharmacological compounds to the red or near-infrared wavelength domain, in which light penetrates human tissue optimally. However, TTA-UC is sensitive to dioxygen, which quenches the triplet states needed for upconversion. Here, we demonstrate not only that the sensitivity of TTA-UC liposomes to dioxygen can be circumvented by adding antioxidants, but also that this strategy is compatible with the activation of ruthenium-based chemotherapeutic compounds. First, red-to-blue upconverting liposomes were functionalized with a blue-light sensitive, membrane-anchored ruthenium polypyridyl complex, and put in solution in presence of a cocktail of antioxidants composed of ascorbic acid and glutathione. Upon red light irradiation with a medical grade 630 nm PDT laser, enough blue light was produced by TTA-UC liposomes under air to efficiently trigger full activation of the Ru-based prodrug. Then, the blue light generated by TTA-UC liposomes under red light irradiation (630 nm, 0.57 W/cm²) through different thicknesses of pork or chicken meat was measured, showing that TTA-UC still occurred even beyond 10 mm of biological tissue. Overall, the rate of activation of the ruthenium compound in TTA-UC liposomes using either blue or red light (1.6 W/cm²) through 7 mm of pork fillet were found comparable, but the blue light caused significant tissue damage, whereas red light did not. Finally, full activation of the ruthenium prodrug in TTA-UC liposomes was obtained under red light irradiation through 7 mm of pork fillet, thereby underlining the in vivo applicability of the activation-by-upconversion strategy.