Photosensitized Singlet Oxygen Production Research Articles

Phosphorous tetrabenzocorrolazine from its metal-free phthalocyanine precursor: Its facile synthesis, high fluorescence emission, efficient singlet oxygen formation, and promising hole transporting material

We have synthesized tetra(4-tert-butyl phenoxy)-tetrabenzocorrolazine phosphorous POTBC(β-OPB)4, a functional π-conjugated macrocycle compound derived from corrole or corrolazine, by a one step procedure from its metal free phthalocyanine precursor. The product structure is characterized by UV–Vis absorption, MS, NMR and IR spectral methods. We have also studied their fluorescence properties, excited triplet state formation, photosensitized singlet oxygen production, and their application as undoped hole transporting layer (HTL) in perovskite solar cells (PSC). The results show that POTBC(β-OPB)4 is highly emissive (fluorescence quantum yield 0.60 in the red region which is little affected by solvent), it also exhibits significant singlet oxygen formation ability (quantum yield 0.40 which is little influenced by solvent), and show remarkable power conversion efficiency (15.8%) as undoped HTL. These facts suggest that POTBC(β-OPB)4 is a promising fluorescence imaging agent and good singlet oxygen photosensitizer for photodynamic therapy of tumor. It is also a good candidate as undoped hole transporting material for PSC.

Tuning photosensitized singlet oxygen production from microgels synthesized by polymerization in aqueous dispersed media

Miniemulsion copolymerization of vinyl acetate, N-vinylcaprolactam, vinyl benzyl Rose Bengal and divinyl adipate to synthesize switchable photosensitizer-grafted polymer colloids for interfacial photooxygenation reactions.

Sulfanyl porphyrazines: Molecular barrel-like self-assembly in crystals, optical properties and in vitro photodynamic activity towards cancer cells

Novel sulfanyl porphyrazines with peripheral 4-bromobenzyl and 4-biphenylylmethyl substituents were synthesized, characterized by photochemical methods and evaluated as sensitizers for photodynamic therapy (PDT). The X-ray crystallography study performed for sulfanyl porphyrazine with 4-biphenylylmethyl substituents revealed that the biphenylyl residues from two consecutive molecules form barrel-like cages with a macrocyclic core constituting a bottom and a top of the barrel and pyridine molecules enclosed inside. The optical properties of both porphyrazines were evaluated in various protic and aprotic solvents. To complement the conventional fluorescence measurements excitation-emission maps were recorded. The potential photosensitizing efficiency of the novel sulfanyl porphyrazines was evaluated by assessing the quantum yields of photosensitized singlet oxygen production. For this purpose the emission of light specific for the transition of singlet oxygen to ground state oxygen was measured. Photodynamic activities of porphyrazines and their liposomal formulations with different surface charge toward two oral squamous cell carcinoma cells (CAL 27, HSC-3) and human cervical epithelial adenocarcinoma cells (HeLa) were determined. Sulfanyl porphyrazines incorporated in cationic liposomes showed high activity, in contrast to the lack of activity of the free form porphyrazines in solution. Thus, cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine can be considered as a promising drug delivery system for the sulfanyl porphyrazines for the photodynamic therapy of cancers.

Solvent dependent photosensitized singlet oxygen production from an Ir(III) complex: pointing to problems in studies of singlet-oxygen-mediated cell death.

A cationic cyclometallated Ir(III) complex with 1,10-phenanthroline and 2-phenylpyridine ligands photosensitizes the production of singlet oxygen, O2(a(1)Δ(g)), with yields that depend appreciably on the solvent. In water, the quantum yield of photosensitized O2(a(1)Δ(g)) production is small (ϕ(Δ) = 0.036 ± 0.008), whereas in less polar solvents, the quantum yield is much larger (ϕ(Δ) = 0.54 ± 0.05 in octan-1-ol). A solvent effect on ϕ(Δ) of this magnitude is rarely observed and, in this case, is attributed to charge-transfer-mediated processes of non-radiative excited state deactivation that are more pronounced in polar solvents and that kinetically compete with energy transfer to produce O2(a(1)Δ(g)). A key component of this non-radiative deactivation process, electronic-to-vibrational energy transfer, is also manifested in pronounced H2O/D2O isotope effects that indicate appreciable coupling between the Ir(III) complex and water. This Ir(III) complex is readily incorporated into HeLa cells and, upon irradiation, is cytotoxic as a consequence of the O2(a(1)Δ(g)) thus produced. The data reported herein point to a pervasive problem in mechanistic studies of photosensitized O2(a(1)Δ(g))-mediated cell death: care must be exercised when interpreting the effective cytotoxicity of O2(a(1)Δ(g)) photosensitizers whose photophysical properties depend strongly on the local environment. Specifically, the photophysics of the sensitizer in bulk solutions may not accurately reflect its intracellular behavior, and the control and quantification of the O2(a(1)Δ(g)) "dose" can be difficult in vivo.

Oxygen‐Dependent Photochemistry and Photophysics of “MiniSOG,” a Protein‐Encased Flavin

Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so-called "mini Singlet Oxygen Generator" (miniSOG), an FMN-containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (ϕΔ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron-transfer reactions (Type-I chemistry) at the expense of photosensitized singlet oxygen production (Type-II chemistry) and results in a comparatively poor yield of singlet oxygen (ϕΔ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well-characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.

Synthesis of Highly Luminescent and Anion-Exchangeable Cerium-Doped Layered Yttrium Hydroxides for Sensing and Photofunctional Applications

The discovery of new lanthanide properties in layered rare-earth hydroxides is tremendously important to developing novel materials with the advantages of both lanthanides and layered hydroxides. Herein, a polyethylenimine-assisted hydrothermal route for preparing Ce-doped layered yttrium hydroxide nanoplates (LYH:Ce NPs) is established, in which the Ce doping provides simultaneous control of the size and fluorescent properties. Typically, 10% Ce doping tailors the average particle size from 680 to 196 nm and induces bright blue luminescence with a quantum efficiency of over 10.0%. Owing to the much more efficient f–d Ce3+ transition, the LYH:Ce NPs show three orders of magnitude stronger photoluminescence than LYH:Eu and LYH:Tb NPs, and exhibit the properties required to fabricate switched “on/off” optical sensors by controlling the Ce3+ Ce4+ redox couple. Furthermore, by combining the merits of the luminescence of Ce3+ dopants and anion-exchange ability of an LYH host, the LYH:Ce NPs exhibit the properties necessary for photofunctional materials for photosensitized singlet oxygen production.

Two-photon induced collagen cross-linking in bioartificial cardiac tissue

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

The role of humic acid aggregation on the kinetics of photosensitized singlet oxygen production and decay

The effect of humic acid (HA) aggregate formation on the photosensitized generation and subsequent quenching of singlet molecular oxygen O(2)(a(1)Δ(g)) was investigated. Time-resolved O(2)(a(1)Δ(g)) phosphorescence traces were obtained from (a) bulk samples of HA dispersions and (b) microscope-based experiments performed upon irradiation of a single HA aggregate. In the bulk experiments, the dependence of the O(2)(a(1)Δ(g)) lifetime on the HA concentration yields a critical concentration for the formation of micrometric HA aggregates of 0.58 g L(-1). This value is consistent with that obtained using pyrene as a fluorescent probe (0.38 g L(-1)). Microscope-based experiments were also performed with HA samples containing added singlet oxygen sensitizers; either the hydrophobic meso-tetraphenylporphyrin (TPP) or the hydrophilic 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP). Singlet oxygen phosphorescence could only be detected upon irradiation of TMPyP, a molecule which localizes on the exterior part of the HA aggregates. The inability to detect O(2)(a(1)Δ(g)) phosphorescence from HA samples containing TPP is consistent with the model that the O(2)(a(1)Δ(g)) produced in the interior of the aggregate was completely quenched by the high local concentration of HA reactive groups in this environment.

Singlet Oxygen Sensor Green®: Photochemical Behavior in Solution and in a Mammalian Cell

The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green(®) (SOSG), a commercially available fluorescent probe for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell. However, for a number of reasons, caution must be exercised when using SOSG. First, it is shown that the immediate product of the reaction between SOSG and singlet oxygen is, itself, an efficient singlet oxygen photosensitizer. Second, SOSG appears to efficiently bind to proteins which, in turn, can influence uptake by a cell as well as behavior in the cell. As such, incorrect use of SOSG can yield misleading data on yields of photosensitized singlet oxygen production, and can also lead to photooxygenation-dependent adverse effects in the system being investigated.

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

Fluorescent proteins are increasingly becoming actuators, rather than just sensors, in a range of cell biology techniques. One of those techniques is chromophore-assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage [1]. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is very useful to avoid uncontrolled photodamage. Indeed, fluorescent proteins have been successfully used in CALI, although of the inactivation mechanisms by ROS are dependent on the fluorescent protein used and are not fully understood [2,3]. Here, we present a quantitative study of the ability of TagRFP to produce ROS, in particular singlet oxygen. TagRFP is able to photosensitize singlet oxygen with an estimated quantum yield of 0.004 [4]. This is the first estimation of a quantum yield of singlet oxygen production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime, which reflects relatively high oxygen accessibility to the chromophore compared to EGFP. Our results provide photophysical insight that allows the understanding of the mechanism behind CALI. Moreover, it has implications in improving photobleaching in fluorescent proteins. [1] K. Jacobson, Z. Rajfur, E. Vitriol, K. Hahn, Trends Cell Biol.2008, 18, 443. [2] M. A. McLean, Z. Rajfur, Z. Z. Chen, D. Humphrey, B. Yang, S. G. Sligar, K. Jacobson, Anal. Chem. 2009, 81, 1755. [3] M. E. Bulina et al, Nat .Biotechnol.2006, 24, 95..[4] X. Ragas, L. P. Cooper, J. H. White, S. Nonell, C. Flors, submitted.

Cover Picture: Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein (ChemPhysChem 1/2011)

Cover Picture: Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein (ChemPhysChem 1/2011)

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

Fluorescent proteins are increasingly becoming actuators in a range of cell biology techniques. One of those techniques is chromophore-assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is useful to avoid uncontrolled photodamage, although the inactivation mechanisms by ROS are dependent on the fluorescent protein and are not fully understood. Herein, we present a quantitative study of the ability of the red fluorescent protein TagRFP to produce ROS, in particular singlet oxygen ((1)O(2)). TagRFP is able to photosensitize (1)O(2) with an estimated quantum yield of 0.004. This is the first estimation of a quantum yield of (1)O(2) production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime compared to EGFP, which reflects relatively high oxygen accessibility to the chromophore. The insight into the structural and photophysical properties of TagRFP has implications in improving fluorescent proteins for fluorescence microscopy and CALI.

Effect of intracellular photosensitized singlet oxygen production on the electrophysiological properties of cultured rat hippocampal neurons

Whole-cell patch-clamp recordings from single cultured mammalian neurons have been used to provide insight into early membrane-dependent events that result upon the intracellular photosensitized production of singlet molecular oxygen, O(2)(a(1)Δ(g)). The singlet oxygen sensitizers used, pyropheophorbide a (PPa) and protoporphyrin IX (PpIX), locate mainly in cell membranes and mitochondria, respectively. Irradiation of these sensitizers altered both passive and dynamic electrophysiological properties of the neurons in a dose-dependent manner, though the response threshold was much lower with PPa than with PpIX. In particular, notable decreases were observed in the rising and falling rates of action potentials and, at higher light fluences, plateau potentials consistent with activation of Ca(2+) channels also developed. The data suggest that singlet oxygen production specifically influences Na(+), K(+) and Ca(2+) ionophores in the cell membrane. Upon terminating sensitizer irradiation, responses evoked by PPa stabilized immediately whereas those evoked by PpIX continued to develop. These data are consistent with a spatially-resolved sphere of intracellular singlet oxygen activity. While the response to PPa irradiation appears to be membrane specific, the response to PpIX irradiation appears to be systemic and possibly part of a cascade of apoptotic events. These results should contribute to a better understanding of membrane-dependent events pertinent to cell death mediated by singlet oxygen.

One- and Two-Photon Photosensitized Singlet Oxygen Production: Characterization of Aromatic Ketones as Sensitizer Standards

Singlet molecular oxygen, O2(a1Deltag), can be efficiently produced in a photosensitized process using either one- or two-photon irradiation. The aromatic ketone 1-phenalenone (PN) is an established one-photon singlet oxygen sensitizer with many desirable attributes for use as a standard. In the present work, photophysical properties of two other aromatic ketones, pyrene-1,6-dione (PD) and benzo[cd]pyren-5-one (BP), are reported and compared to those of PN. Both PD and BP sensitize the production of singlet oxygen with near unit quantum efficiency in a nonpolar (toluene) and a polar (acetonitrile) solvent. With their more extensive pi networks, the one-photon absorption spectra for PD and BP extend out to longer wavelengths than that for PN, thus providing increased flexibility for sensitizer excitation over the range approximately 300-520 nm. Moreover, PD and BP have much larger two-photon absorption cross sections than PN over the range 655-840 nm which, in turn, results in amounts of singlet oxygen that are readily detected in optical experiments. One- and two-photon absorption spectra of PD and BP obtained using high-level calculations model the salient features of the experimental data well. In particular, the ramifications of molecular symmetry are clearly reflected in both the experimental and calculated spectra. The use of PD and BP as standards for both the one- and two-photon photosensitized production of singlet oxygen is expected to facilitate the development of new sensitizers for application in singlet-oxygen-based imaging experiments.

Control and Selectivity of Photosensitized Singlet Oxygen Production: Challenges in Complex Biological Systems

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Effect of Sensitizer Protonation on Singlet Oxygen Production in Aqueous and Nonaqueous Media

The yield of singlet molecular oxygen, O2(a(1)Delta(g)), produced in a photosensitized process can be very susceptible to environmental perturbations. In the present study, protonation of photosensitizers whose chromophores contain amine functional groups is shown to adversely affect the singlet oxygen yield. Specifically, for bis(amino) phenylene vinylenes dissolved both in water and in toluene, addition of a protic acid to the solution alters properties of the system that, in turn, result in a decrease in the efficiency of singlet oxygen production. In light of previous studies on other molecules where protonation-dependent changes in the yield of photosensitized singlet oxygen production have been ascribed to changes in the quantum yield of the sensitizer triplet state, Phi(T), and to possible changes in the triplet state energy, E(T), our results demonstrate that this photosystem can respond to protonation in other ways. Although protonation-dependent changes in the amount of charge-transfer character in the sensitizer-oxygen complex may influence the singlet oxygen yield, it is likely that other processes also play a role. These include (a) protonation-dependent changes in sensitizer aggregation and (b) nonradiative channels for sensitizer deactivation that are enhanced as a consequence of the reversible protonation/deprotonation of the chromophore. The data obtained, although complicated, are relevant for understanding and ultimately controlling the behavior of photosensitizers in systems with microheterogeneous domains that have appreciable pH gradients. These data are particularly important given the use of such bi-basic chromophores as two-photon singlet oxygen sensitizers, with applications in spatially resolved singlet oxygen experiments (e.g., imaging experiments).

Control and Selectivity of Photosensitized Singlet Oxygen Production: Challenges in Complex Biological Systems

Singlet molecular oxygen is a reactive oxygen species that plays an important role in a number of biological processes, both as a signalling agent and as an intermediate involved in oxidative degradation reactions. Singlet oxygen is commonly generated by the so-called photosensitization process wherein a light-absorbing molecule, the sensitizer, transfers its energy of excitation to ground-state oxygen to make singlet oxygen. This process forms the basis of photodynamic therapy, for example, where light, a sensitizer, and oxygen are used to initiate cell death and ultimately destroy undesired tissue. Although the photosensitized production of singlet oxygen has been studied and used in biologically pertinent systems for years, the photoinitiated behaviour is often indiscriminate and difficult to control. In this Concept, we discuss new ideas and results in which spatial and temporal control of photosensitized singlet oxygen production can be implemented through the incorporation of the sensitizer into a conjugate system that selectively responds to certain triggers or stimuli.

Triplet state properties of N-mTEG[60]fulleropyrrolidine mono and bisadduct derivatives

Laser flash photolysis and pulse radiolysis techniques were used to determine triplet state properties of N-mTEG[60]fulleropyrrolidine mono and bisadduct derivatives (FP) (mTEG=CH 2CH 2OCH 2CH 2OCH 2CH 2OCH 3) in benzene. These properties include the triplet absorption spectra between 550 and 900 nm, quantum yields of triplet photosensitized singlet oxygen production ( Φ Δ ) and triplet molar absorption coefficients ( ε T) with the assumption that the quantum yield of triplet formation ( Φ T) is equal to Φ Δ . Our results demonstrate that the triplet properties depend on the number of addends and the addition pattern.

Quantum Yield of Photosensitized Singlet Oxygen (a1.DELTA.g) Production in Solid Polystyrene

The quantum yield of singlet oxygen (a[sup 1][Delta][sub g]), produced by energy transfer from the photosensitizer acridine, has been determined by two independent spectroscopic methods in solid polystyrene. Upon 355-nm pulsed-laser irradiation of acridine at 1.3 mJ/pulse, the O[sub 2](a[sup 1][Delta][sub g]) quantum yield in polystyrene [[phi][sub [Delta]][sup PS](acridine) = 0.56 [+-] 0.05] is slightly smaller than that in a liquid solvent analog such as benzene [[phi][sub [Delta]][sup benz](acridine) = 0.83 [+-] 0.06]. These data indicate that the photosensitized yields of O[sub 2](a[sup 1][Delta][sub g]) in a solid polymer matrix can nevertheless be appreciable, which may be important for understanding the photodegradation of some polymeric materials or dyes and other additives dissolved in those materials.

Porphyrin photosensitization and phototherapy.

Porphyrin photosensitization and phototherapy.