Highlighting the Phototherapeutical Potential of Fungal Pigments in Various Fruiting Body Extracts with Informed Feature-Based Molecular Networking.

Fungal pigments are characterized by a diverse set of chemical backbones, some of which present photosensitizer-like structures. From the genus Cortinarius, for example, several biologically active photosensitizers have been identified leading to the hypothesis that photoactivity might be a more general phenomenon in the kingdom Fungi. This paper aims at testing the hypothesis. Forty-eight fruiting body-forming species producing pigments from all four major biosynthetic pathways (i.e., shikimate-chorismate, acetate-malonate, mevalonate, and nitrogen heterocycles) were selected and submitted to a workflow combining in vitro chemical and biological experiments with state-of-the-art metabolomics. Fungal extracts were profiled by high-resolution mass spectrometry and subsequently explored by spectral organization through feature-based molecular networking (FBMN), including advanced metabolite dereplication techniques. Additionally, the photochemical properties (i.e., light-dependent production of singlet oxygen), the phenolic content, and the (photo)cytotoxic activity of the extracts were studied. Different levels of photoactivity were found in species from all four metabolic groups, indicating that light-dependent effects are common among fungal pigments. In particular, extracts containing pigments from the acetate-malonate pathway, e.g., extracts from Bulgaria inquinans, Daldinia concentrica, and Cortinarius spp., were not only efficient producers of singlet oxygen but also exhibited photocytotoxicity against three different cancer cell lines. This study explores the distribution of photobiological traits in fruiting body forming fungi and highlights new sources for phototherapeutics.

A review of pesticide phototransformation on the leaf surface: Models, mechanism, and influencing factors.

Phototransformation is an important environmental fate of pesticides on plant leaves. This review found that the photodegradation rates of pesticides on leaves might be faster or slower than those in organic solvents or on glass because of the different spectral patterns and light fluxes on the model surface. Wax was found to play an important role in pesticide phototransformation because it has photosensitizing properties, which might be stimulated under light irradiation to produce reactive species, such as hydroxyl radicals, singlet oxygen, methyl radicals, alkyl radicals, and superoxide radicals. These reactive species could accelerate pesticide photodegradation by several times. Wax can also decrease the photodegradation rate of pesticides by quenching reactive species or light-shielding effects. The environmental conditions and phytochemical properties of leaves play important roles in pesticide phototransformation primarily because the composition of wax varies with plant species and environmental factors. The phototransformation of pesticides on leaves was promoted by a low dosage of adjuvant because they act as photosensitizers and improve the dispersity of pesticides, while it was inhibited at a high concentration of adjuvant because of their light shielding effect. Finally, recommendations for future research were discussed, including (1) distinguishing the direct and indirect photodegradation of pesticides; (2) developing model, molecular level visualization and analysis techniques; (3) conducting more field research; and (4) considering the effect of climate change, especially the interaction of climatic factors. This review gives a comprehensive overview of the current knowledge of pesticide phototransformation on leaves and provides suggestions for future studies.

Antiproliferative and Cytotoxic Activities of Fluorescein-A Diagnostic Angiography Dye.

Fluorescein is a fluorescent dye used as a diagnostic tool in various fields of medicine. Although fluorescein itself possesses low toxicity, after photoactivation, it releases potentially toxic molecules, such as singlet oxygen (1O2) and, as we demonstrate in this work, also carbon monoxide (CO). As both of these molecules can affect physiological processes, the main aim of this study was to explore the potential biological impacts of fluorescein photochemistry. In our in vitro study in a human hepatoblastoma HepG2 cell line, we explored the possible effects on cell viability, cellular energy metabolism, and the cell cycle. We observed markedly lowered cell viability (≈30%, 75-2400 µM) upon irradiation of intracellular fluorescein and proved that this decrease in viability was dependent on the cellular oxygen concentration. We also detected a significantly decreased concentration of Krebs cycle metabolites (lactate and citrate < 30%; 2-hydroxyglutarate and 2-oxoglutarate < 10%) as well as cell cycle arrest (decrease in the G2 phase of 18%). These observations suggest that this photochemical reaction could have important biological consequences and may account for some adverse reactions observed in fluorescein-treated patients. Additionally, the biological activities of both 1O2 and CO might have considerable therapeutic potential, particularly in the treatment of cancer.

A thermally activated delayed fluorescence photosensitizer for photodynamic therapy of oral squamous cell carcinoma under low laser intensity.

In this report, a new thermally activated delayed fluorescence (TADF) molecule [2-(4-triphenylvinyl-phenyl)-anthraquinone (TPE-AQ)] was synthesized. This nanomaterial has satisfactory photostability. Through In vitro analysis, it was found that these TADF nanoparticles (NPs) targeted lysosomes in oral cancer cells. ROS were released under irradiation with a 450-nm laser, and the growth of xenograft tumors in mouse models was inhibited in vivo. More interestingly, radiation exposure caused little damage to normal tissues due to the low irradiation intensity (mA) used in the photodynamic therapy (PDT) treatment of oral cancer. Therefore, these TADF NPs provide new possibilities for the development of new PDT drugs for biomedical applications. In future work, possible functional modifications of TADF NPs for increased potency in clinical applications will be addressed.

Chlorin e6-1,3-diphenylisobenzofuran polymer hybrid nanoparticles for singlet oxygen-detection photodynamic abaltion.

A dual-functional nanosysterm is developed by means of Chlorin e6 (Ce6) as photosensitizer and 1,3-Diphenylisobenzofuran (DPBF) as fluorescent singlet oxygen (1O2) probe. Under 660 nm laser irradiation, Ce6 exhibites efficient 1O2 generation, and subsequently the production of 1O2 is assessed by the ratiometric fluorescence of PFO and DPBF under one-photon and two-photon excitation mode. The nanoparticles with excellent biocompatibility can be internalized into Hela cells and applied for tumor treatment. For intracellular PDT, the nanoparticles perform a high phototoxicity, while the PDT proccess can be evaluated in time by monitoring fluorescence signals of DPBF. This theranostic nanosysterm provides a facile strategy to fabricate 1O2-detection PDT, which can realize accurate and efficient photodynamic therapy based on singlet oxygen detection.

Bioengineering a Light-Responsive Encapsulin Nanoreactor: A Potential Tool for In Vitro Photodynamic Therapy.

Encapsulins, a prokaryotic class of self-assembling protein nanocompartments, are being re-engineered to serve as "nanoreactors" for the augmentation or creation of key biochemical reactions. However, approaches that allow encapsulin nanoreactors to be functionally activated with spatial and temporal precision are lacking. We report the construction of a light-responsive encapsulin nanoreactor for "on demand" production of reactive oxygen species (ROS). Herein, encapsulins were loaded with the fluorescent flavoprotein mini-singlet oxygen generator (miniSOG), a biological photosensitizer that is activated by blue light to generate ROS, primarily singlet oxygen (1O2). We established that the nanocompartments stably encased miniSOG and in response to blue light were able to mediate the photoconversion of molecular oxygen into ROS. Using an in vitro model of lung cancer, we showed that ROS generated by the nanoreactor triggered photosensitized oxidation reactions which exerted a toxic effect on tumor cells, suggesting utility in photodynamic therapy. This encapsulin nanoreactor thus represents a platform for the light-controlled initiation and/or modulation of ROS-driven processes in biomedicine and biotechnology.

Effects of zinc porphyrin and zinc phthalocyanine derivatives in photodynamic anticancer therapy under different partial pressures of oxygen in vitro.

Photodynamic therapy (PDT) is gradually becoming an alternative method in the treatment of several diseases. Here, we investigated the role of oxygen in photodynamically treated cervical cancer cells (HeLa). The effect of PDT on HeLa cells was assessed by exposing cultured cells to disulphonated zinc phthalocyanine (ZnPcS2) and tetrasulphonated zinc tetraphenylporphyrin (ZnTPPS4). Fluorescence microscopy revealed their different localizations within the cells. ZnTPPS4 seems to be mostly limited to the cytosol and lysosomes, whereas ZnPcS2 is most likely predominantly attached to membrane structures, including plasmalemma and the mitochondrial membrane. Phototoxicity assays of PDT-treated cells carried out under different partial pressures of oxygen showed dose-dependent responses. Interestingly, ZnPcS2 was also photodynamically effective at a minimal level of oxygen, under a nitrogen atmosphere. On the other hand, hyperbaric oxygenation did not lead to a higher PDT efficiency of either photosensitizer. Although both photosensitizers can induce a significant drop in mitochondrial membrane potential, ZnPcS2 has a markedly higher effect on mitochondrial respiration that was completely blocked after two short light cycles. In conclusion, our observations suggest that PDT can be effective even in hypoxic conditions if a suitable sensitizer is chosen, such as ZnPcS2, which can inhibit mitochondrial respiration.

Singlet Oxygen and Protochlorophyllide Detection in Arabidopsis thaliana.

Since the recognition of the reactive oxygen species singlet oxygen (1O2) as a versatile signal that induces various stress responses, the mechanisms underlying 1O2-induced signaling transduction pathways have become the subject of much current research. This in turn highlights the need for reliable detection methods for 1O2. Here we describe a protocol for the detection of 1O2 using a commercially available fluorescent probe (Singlet Oxygen Sensor Green) and provide a simple method for direct visualization and quantification of the 1O2-evolving photosensitizer protochlorophyllide in the Arabidopsis fluorescent mutant.

Methods to Unravel Pathways of Reactive Oxygen Species in the Photodynamic Inactivation of Bacteria.

Different experimental conditions can be used to detect the presence of reactive oxygen species (ROS) in the photodynamic inactivation of microorganisms. Here, we describe the effect of the media and the addition of ROS scavengers to obtain insight about the oxidative processes that take place during the photokilling of bacteria. In addition, 9,10-dimethylanthracene was used to sense the generation of singlet molecular oxygen, O2(1Δg), in microbial cells. Thus, the contribution of type I or type II pathways in the photocytotoxicity action can be rapidly detected and compared between different photosensitizers.

Revealing ROS Production by Antibiotics and Photosensitizers in Biofilms: A Fluorescence Microscopy Approach.

Reactive oxygen species (ROS) production within biofilms is studied with a simple and easy setup based on fluorescence microscopy. Herein, a biofilm is exposed to different ROS inducers: a bactericidal antibiotic (ciprofloxacin) and a BODIPY-based photosensitizer (I2B-OAc). Real-time ROS induction in the core of the biofilms is monitored utilizing two fluorescent reporters-AMDA and H2DCFDA-the first one with selectivity toward singlet oxygen (1O2) and the latest for other ROS (O2•-, H2O2, and OH•-). A point-by-point methodology is reported, starting with the sample preparation all the way through the microscope setup and, finally, processing of the images.

Singlet Oxygen Quantum Yield Determination Using Chemical Acceptors.

Singlet oxygen (1O2) is the first electronic excited state of molecular oxygen. Due to its non-radical and non-ionic character as well as its mild reactivity, 1O2 has a pivotal role in cell signaling processes at low concentration, yet it is cytotoxic at high concentrations. Quantifying the production of 1O2, particularly in biological systems, is therefore essential for understanding and controlling its effects. 1O2 can be produced by chemical and biological reactions, yet its most common method of production is by photosensitization, whereby an initially photoexcited molecule transfers its acquired electronic energy to the dioxygen molecule. The efficiency of this process is characterized by the 1O2 production quantum yield, ΦΔ, which can be determined by directly monitoring its intrinsic weak near-infrared phosphorescence or indirectly by trapping it with a suitable acceptor, a process that can be monitored by common analytical techniques. Indirect methods are thus very popular, yet they may lead to severe errors if used incorrectly. Herein we describe the common aspects of indirect methods and propose a general step-by-step procedure for the determination of ΦΔ values. In addition, we identify the key experimental conditions that need to be controlled to obtain meaningful results.

A General Method to Establish the Relative Efficiency of Different Sonosensitizers to Generate ROS for SDT.

The most common way to demonstrate the reactive oxygen species (ROS)-mediated pathways in photodynamic therapy (PDT) and in sonodynamic therapy (SDT) is the use of specific ROS inhibitors. We present a general method to establish the relative efficiency of different sonosensitizers which produce the same ROS. To demonstrate it, we use peroxides as sonosensitizers which produce singlet molecular oxygen. The method is easily generalized by all types of ROS.

A fluorous sodium l-prolinate derivative as low molecular weight gelator for perfluorocarbons.

We report the first study dealing with the self-assembly of an α-amino acid derivative in perfluorocarbons. Rheology, microscopy, and spectroscopy studies reveal that the fluorous sodium l-prolinate derivative 1 self-assembles in perfluorocarbons to form a three-dimensional network of left-handed nano-helices resulting in solvent gelation. Singlet oxygen lifetime measured in a gel of perfluorodecalin is about 1000 times longer than in pure water.

Palladium porphyrin complexes for photodynamic cancer therapy: effect of porphyrin units and metal.

Photodynamic therapy (PDT) has been extensively explored for malignant tissue treatment. In this work, we successfully synthesized and characterized a series of porphyrin compounds by connecting porphyrin units with alkyl chains, which were then coordinated with palladium to yield related metal complexes, named Pd-Monopor, Pd-Dipor, and Pd-Tripor, respectively. The generation of reactive oxygen species (ROS) of six porphyrin compounds was investigated by the dichlorofluorescein (DCFH) method. As expected, the palladium porphyrin complexes showed the higher efficiency of ROS generation relative to free base porphyrins, probably due to the heavy atom effect. Remarkably, the efficiency of ROS generation increased with the number of porphyrin units in the photosensitizers. The order of ROS generation efficiency of the synthesized porphyrins was Pd-Tripor > Tripor > Dipor > Pd-Monopor > Pd-Dipor > Monopor. MTT assay suggested the good biocompatibility of the synthesized photosensitizers in the dark. Upon light irradiation, the palladium porphyrin complex exhibited higher therapeutic activity than free base porphyrin. The half-maximal inhibitory concentration (IC50) of Tripor and Pd-Tripor under light irradiation was calculated to be 18.2 and 9.6 µM, respectively. The cellular uptake and subcellular localization experiments indicated that Tripor was mainly localized in the lysosomes of cancer cells.

Carbon dot-assisted luminescence of singlet oxygen: the generation dynamics but not the cumulative amount of singlet oxygen is responsible for the photodynamic therapy efficacy.

A novel carbon dot-based luminescence probe for singlet oxygen (1O2) with a conventional optical detector has been implemented through the specific formation of electronically excited carbonyls from the breakdown of unstable endoperoxide intermediates, and its application in the real-time in vivo monitoring of 1O2 in photodynamic therapy (PDT) is achieved. More attractively, the relationship between the dynamics details of photosensitizer-generated 1O2 and the PDT efficacy has been established through a modified multiple-target survival model, enabling a direct and easy estimate of the surviving fraction of tumor cells from the generation dynamics of 1O2. Both in vitro and in vivo therapy results revealed that the rapid generation dynamics of 1O2 rather than its cumulative amount is responsible for better treatment efficacy in PDT. Overall, the deeper insight into the important roles of the generation dynamics of 1O2 in the PDT efficacy is irreplaceably advantageous in substantially reduced risks from deleterious treatment-related side effects by screening advanced photosensitizers and determining the light exposure end point.

Light-assisted gadofullerene nanoparticles disrupt tumor vasculatures for potent melanoma treatment.

The traditional photodynamic therapy (PDT) using a photosensitizer and oxygen under light generates reactive oxygen species (ROS) to kill tumor cells. However, its treatment efficiency is limited by insufficient oxygen in tumor cells. Herein, ß-alanine modified gadofullerene nanoparticles (GFNPs) were explored to disrupt tumor vasculatures assisted by light for potent melanoma treatment. As tumor vasculatures are oxygen-rich, the yields of photo-induced singlet oxygen (1O2) by GFNPs are not subjected to the hypoxemia of tumor tissues. Different from the small molecule photosensitizer Chlorin e6 (Ce6), GFNPs realize high-efficiency tumor vascular disruption under light observed by using the mice tumor vascular dorsal skin fold chamber (DSFC) model. The tumor vascular disruption efficiency of GFNPs is size-dependent, and the smallest one (hydration diameter of ca. 126 nm) is more efficient. Mechanistically, the high yields of photo-induced 1O2 by GFNPs can lead to the destruction of the tumor vascular endothelial adherent junction protein-VE cadherin and the decrease of tumor vascular endothelial cells-CD31 proteins, inducing rapid tumor necrosis. In conclusion, our work provides an insight into the design of well-sized nanoparticles to powerfully treat melanoma assisted by light, as well as greatly extending the applications of PDT for robust tumor therapy.

Basic Investigations of Singlet Oxygen Detection Systems with ESR for the Measurement of Singlet Oxygen Quenching Activities.

Singlet oxygen (1O2) is highly oxidative and exerts strong cytotoxic effects. We tried to establish the best combination of a singlet oxygen generation system and a detection method with ESR, for measurement of the quenching activities of various substances. The photosensitizing reaction of rose bengal or thermal decomposition of 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide, EP) was used for the generation of 1O2, and a sterically hindered secondary amine, 2,2,6,6-tetramethyl-4-piperidone (TEMPD) or 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH), was used as the 1O2 detection probe. These secondary amines were oxidized by 1O2 to form stable nitroxide radicals, which were detectable by ESR. TEMPD was found to be readily oxidized by air, causing large background signals in comparison with TEMP-OH. The ESR signal obtained by the irradiation of rose bengal with visible light in the presence of TEMP-OH consisted of two kinds of nitroxide radical overlapping. In contrast, only a single nitroxide signal was observed when TEMP-OH was reacted with 1O2 generated from EP. Therefore, the best combination should be EP as the 1O2 generator and TEMP-OH as the detection probe. When using this combination, we found that the concentrations of some organic solvents such as dimethyl sulfoxide and acetonitrile should be kept constant for reliable quantification, because the concentrations of organic solvents affect the ESR signal intensity.

The detection sensitivity of commonly used singlet oxygen probes in aqueous environments.

The sensitivity for singlet oxygen (1O2) of two convenient 1O2 probes, 1,3-diphenylisobenzofuran (DPBF) and 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA), has been investigated in different aqueous environments. Both probes are commercially available at reasonable cost and can be used with standard UV-vis spectrometers. Although DPBF is not soluble in neat water and is not specific to the detection of 1O2, it has very high, essentially diffusion-limited, reactivity towards 1O2; it can trap up to 50% of all 1O2 created in alcohol/water or micellar solution, and even more when replacing H2O by D2O, which makes it highly useful when the process under investigation does not yield much 1O2. On the other hand, ABDA has a much lower reactivity, reacting with only 2% of the singlet oxygen generated in H2O, as well as a smaller extinction coefficient, resulting in a much smaller spectroscopic response, but is soluble in neat water and is specific for 1O2, allowing for discrimination from other reactive oxygen species. The results presented here not only allow a comparative assessment of the usefulness of the two 1O2 probes, but also provide a reference for an accurate absolute quantification of the amount of 1O2 generated in an experiment from the observed absorbance bleach.

Post-synthesis strategy to integrate porphyrinic metal-organic frameworks with CuS NPs for synergistic enhanced photo-therapy.

Multifunctional nanotheranostic systems with both therapeutic and imaging functions are highly desired for the development of more effective and less toxic anti-tumor drugs. Herein, a simple but effective method is reported to fabricate a novel PCN-CuS-FA-ICG-based nanoplatform for dual-modal imaging-guided synergistic photothermal/photodynamic therapy. Porphyrinic metal-organic frameworks with CuS NPs are obtained in aqueous solution via a simple post-synthesis strategy. Furthermore, to obtain a more effective therapy, indocyanine green (ICG) was incorporated into the multifunctional theranostic platform to promote the photothermal therapeutic effect. The as-prepared PCN-CuS-FA-ICG not only exhibits an excellent 1O2 generation efficiency under 650 nm irradiation to achieve remarkable photodynamic cell killing, but also presents outstanding photothermal conversion under 808 nm irradiation to destroy tumor tissues by hyperthermia. In particular, the nanotherapeutic agent realized fluorescence and thermal imaging dual-modal imaging-guided cancer treatment. Meanwhile, in vivo experiments confirmed the evident accumulation of nanoparticles (NPs) at local tumors, and tumor growth was inhibited obviously via synergistic photothermal/photodynamic therapy with negligible side effects.

1O2 determined from the measured PDT dose and 3O2 predicts long-term response to Photofrin-mediated PDT.

Photodynamic therapy (PDT) that employs the photochemical interaction of light, photosensitizer and oxygen is an established modality for the treatment of cancer. However, dosimetry for PDT is becoming increasingly complex due to the heterogeneous photosensitizer uptake by the tumor, and complicated relationship between the tissue oxygenation ([3O2]), interstitial light distribution, photosensitizer photobleaching and PDT effect. As a result, experts argue that the failure to realize PDT's true potential is, at least partly due to the complexity of the dosimetry problem. In this study, we examine the efficacy of singlet oxygen explicit dosimetry (SOED) based on the measurements of the interstitial light fluence rate distribution, changes of [3O2] and photosensitizer concentration during Photofrin-mediated PDT to predict long-term control rates of radiation-induced fibrosarcoma tumors. We further show how variation in tissue [3O2] between animals induces variation in the treatment response for the same PDT protocol. PDT was performed with 5 mg kg-1 Photofrin (a drug-light interval of 24 h), in-air fluence rates (ϕ air) of 50 and 75 mW cm-2 and in-air fluences from 225 to 540 J cm-2. The tumor regrowth was tracked for 90 d after the treatment and Kaplan-Meier analyses for local control rate were performed based on a tumor volume ⩽100 mm3 for the two dosimetry quantities of PDT dose and SOED. Based on the results, SOED allowed for reduced subject variation and improved treatment evaluation as compared to the PDT dose.