Targeted isolation of photoactive pigments from mushrooms yielded a highly potent new photosensitizer: 7,7'-biphyscion.

Pigments of fungi are a fertile ground of inspiration: they spread across various chemical backbones, absorption ranges, and bioactivities. However, basidiomycetes with strikingly colored fruiting bodies have never been explored as agents for photodynamic therapy (PDT), even though known photoactive compound classes (e.g., anthraquinones or alkaloids) are used as chemotaxonomic markers. In this study, we tested the hypothesis that the dyes of skin-heads (dermocyboid Cortinarii) can produce singlet oxygen under irradiation and thus are natural photosensitizers. Three photosensitizers based on anthraquinone structures were isolated and photopharmaceutical tests were conducted. For one of the three, i.e., (-)-7,7'-biphyscion (1), a promising photoyield and photocytotoxicity of EC50 = 0.064 µM against cancer cells (A549) was found under blue light irradiation (λexc = 468 nm, 9.3 J/cm2). The results of molecular biological methods, e.g., a viability assay and a cell cycle analysis, demonstrated the harmlessness of 1 in the dark and highlighted the apoptosis-inducing PDT potential under blue light irradiation. These results demonstrate for the first time that pigments of dermocyboid Cortinarii possess a so far undescribed activity, i.e., photoactivity, with significant potential for the field of PDT. The dimeric anthraquinone (-)-7,7'-biphyscion (1) was identified as a promising natural photosensitizer.

The Cosmetic Potential of The Medicinal Halophyte Tamarix Gallica L. (Tamaricaceae) Growing in The Eastern Part of Algeria: Photoprotective and Antioxidant Activities.

AIM AND OBJECTIVE: Currently, the use of ingredients from natural sources has gained great attention in the cosmetic field, especially for the development of new photoprotective formulations. Therefore, the present study aimed to evaluate the cosmetic potential of the crude methanol extract and the ethyl acetate fraction of the medicinal halophyte Tamarix gallica L. (Tg) growing in the area of Tebessa in the eastern part of Algeria, by assessing their phenolic and flavonoid contents, photoprotective and antioxidant activities. METHODS: The research approach consisted of determining phenolic and flavonoid contents of aerial parts via Folin-Ciocalteu and aluminum chloride methods, respectively. The antioxidant activity was measured through two in vitro methods, DPPH radical scavenging activity and total antioxidant capacity test (TAC). The in vitro photoprotective effect was evaluated according to the parameter SPF (Sun Protection Factor) by using the UV spectroscopic method in the UV-B region (290-320 nm). RESULTS: The methanol extract (Tg-MeOH) and ethyl acetate (Tg-EtOAc) fraction showed good antioxidant activity with IC50 values of 14.05±0.66, 27.58±1.98 µg/mL respectively in the DPPH test. Furthermore, both extracts displayed strong total antioxidant capacity (287.01±7.85, 246.7±1.12 mg AAE/g, respectively) in the TAC test. Both extracts exhibited high photoprotective activity, with sun protection factor (SPF) values 37.03±0.22 and 36.08±0.03. The antioxidant and photoprotective activities of these extracts were probably related to polyphenols content (190.27±0.74 mg AGE /g and 121.77±1.29 mg AGE /g, respectively) and flavonoids (78.75±0.06 mg QE /g and 58.67±1.19mg/g). CONCLUSION: Our findings show that extracts of Tamarix gallica L. could be a promising source to be mixed as a natural sunscreen and antioxidant agents into photoprotective cosmetic formulations.

Photosensitizing Furocoumarins: Content in Plant Matrices and Kinetics of Supercritical Carbon Dioxide Extraction.

Furocoumarins are a group of plant phytoalexins exhibiting various bioactive properties; the most important of which are photosensitization and alteration of P450 cytochrome activity. Supercritical fluid extraction with carbon dioxide has been proposed as a green alternative for an organic solvent extraction of the furocoumarins. Four plant matrices rich in furocoumarins were extracted with CO2 at a temperature of 80 °C and pressure of 40 MPa, as these conditions were characterized by the highest solubility of furocoumarins. The extracts collected were analyzed using the HPLC method and the results obtained were used for the mathematical modeling of the observed phenomena. The total content of the furocoumarins in the matrices was 4.03-26.45 mg g-1 of dry weight. The impact of the process parameters on the solubility was consistent with the Chrastil equation. The broken plus intact cell model proved to be suitable to describe extraction curves obtained. The research proved the possibility of supercritical carbon dioxide utilization for the extraction of the furocoumarins from plant material and provided valuable data for prospective industrial-scale experiments.

Exploring the Role of Phytochemicals as Potent Natural Photosensitizers in Photodynamic Therapy.

BACKGROUND: Cancer is still considered a deadly disease worldwide due to difficulties in diagnosis, painful treatment procedures, costly therapies, side effects, and cancer relapse. Cancer treatments using conventional methods like chemotherapy and radiotherapy were not convincing due to its post-treatment toxicity in the host. In Photodynamic Therapy (PDT), three individual non-toxic components including a photosensitizer, light source and oxygen cause damage to the cells and tissues when they are combined. OBJECTIVE: In recent years, phytochemicals are being increasingly recognized as potent complementary drugs for cancer because of its natural availability, less toxicity and therapeutic efficiency in par with commercial drugs. Hence, the idea of using phytochemicals as natural photosensitizers in PDT resulted in a multiple pool of research studies with promising results in preclinical and clinical investigations. METHODS: In this review, the potential of phytochemicals to act as natural photosensitizers for PDT, their mode of action, drawbacks, challenges and possible solutions are discussed in detail. RESULTS: In PDT, natural photosensitizers, when used alone or in combination with other photosensitizers, induced cell death by apoptosis and necrosis, increased oxidative stress, altered cancer cell death signaling pathways, increased cytotoxicity and DNA damage in cancer cells. The pro-oxidant nature of certain antioxidant polyphenols, hormesis phenomenon, Warburg effect and DNA damaging potential plays a significant role in the photosensitizing mechanism of phytochemicals in PDT. CONCLUSION: This review explores the role of phytochemicals that can act as photosensitizers alone or in combination with PDT and its mechanism of action on different cancers.

Photodynamic action of Hypericum perforatum hydrophilic extract against Staphylococcus aureus.

Hypericin (Hyp) is one of the most effective, naturally occurring photodynamic agents, which proved effective against a wide array of microorganisms. One limitation of its large scale application as a disinfectant is the high production cost of the pure compound. The availability of photoactive materials at a lower cost may be highly beneficial to the actual implementation of photodisinfection also at the industrial level. In this work we report the use of a lyophilized extract from Hypericum perforatum as a photosensitizing material. We show that optical absorption in the green-red region of the visible spectrum of ethanol or DMSO solutions of the lyophilized extract contains bands arising from Hyp. When excited with light in the main Hyp absorption bands, fluorescence emission and triplet state formation occur as in pure Hyp solutions. We show that ethanol or DMSO solutions of the lyophilized extract from Hypericum perforatum are highly efficient photodynamic agents against Gram-positive Staphylococcus aureus, chosen as a model. The performance is indistinguishable from that of the pure compound. Using fluorescence microscopy, we demonstrate that upon incubation of S. aureus with lyophilized extract solutions, Hyp is found on the bacterial wall, as previously reported for the pure compound.

Datura suaveolens and Verbena tenuisecta mediated silver nanoparticles, their photodynamic cytotoxic and antimicrobial evaluation.

Biogenic production of nanoparticles is eco-friendly, less expensive method with various medical and biological applications. Nanotechnology along with photodynamic therapy is gaining tremendous importance with enhanced efficacy. The present work was aimed to evaluate methanolic extracts and nanoparticles of two selected plants (Datura suavolens and Verbina tenuisecta) for cytotoxic photodynamic, antioxidant and antimicrobial study. Both extract and silver (5 mM) nanoparticles of Datura plant showed significant activities against bacterial strains. Maximum ZOI of 27.3 ± 1.6 mm was observed with nanoparticles of Datura branches with minimum inhibitory (MIC) value of 32 µg/ml. In case of antifungal and antioxidant assay samples were moderately active. Silver nanoparticles and extracts were effective against rhabdomyosarcoma cell line with lowest IC50 value of 42.5 ± 0.6 µg/ml and percent viability of 25.6 ± 1.3 of Verbena tenuisecta. However, nanoparticles of Datura leaves and branches were more potent with IC50 value of 2.4 ± 0.9 µg/ml and 7.8 ± 1.1 µg/ml respectively. The result of photodynamic study showed that efficacy of photosensitizer was enhanced and percent viability reduced when nanoparticles used as an adjunct. The color change and UV spectra (415‒425 nm) indicated the production of nanoparticles. Fourier transform infrared spectroscopy (FTIR) spectra showed presence of different functional groups e.g., hydroxyl, carbonyl and amino. Nanoparticles are sphenoid in morphology and size ranges between 20-150 nm. Current study showed these silver nanoparticles can be used as cytotoxic agent in photodynamic therapy and can play a critical role to establish medicinal potential of selected plants.

A Cytotoxic Porphyrin from North Pacific Brittle Star Ophiura sarsii.

Triple-negative breast cancer (TNBC) represents the deadliest form of gynecological tumors currently lacking targeted therapies. The ethanol extract of the North Pacific brittle star Ophiura sarsii presented promising anti-TNBC activities. After elimination of the inert material, the active extract was submitted to a bioguided isolation approach using high-resolution semipreparative HPLC-UV, resulting in one-step isolation of an unusual porphyrin derivative possessing strong cytotoxic activity. HRMS and 2D NMR resulted in the structure elucidation of the compound as (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid. Never identified before in Ophiuroidea, porphyrins have found broad applications as photosensitizers in the anticancer photodynamic therapy. The simple isolation of a cytotoxic porphyrin from an abundant brittle star species we describe here may pave the way for novel natural-based developments of targeted anti-cancer therapies.

The effects of photodynamic therapy on leukemia cells mediated by KillerRed, a genetically encoded fluorescent protein photosensitizer.

BACKGROUND: Leukemia is a cancer of blood and bone marrow cells, causing about 300,000 deaths worldwide. Photodynamic therapy (PDT) is a promising alternative for the treatment of malignant tumors. KillerRed is a genetically encoded red fluorescent protein photosensitizer (PS). In this study, we aimed to investigate the effects of KillerRed-mediated PDT on chronic myelogenous leukemia K562 cells, acute monocytic leukemia NB4 cells, and acute monocytic leukemia THP1 cells. METHODS: KillerRed was expressed in Escherichia coli cells, purified by Q-Sepharose column, and confirmed by western-blotting. The PDT effect on cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8). Cell apoptosis was determined by PE Annexin V/7-AAD staining and flow cytometry. The distribution of KillerRed in leukemia cells was detected by confocal laser scanning microscopy (CLSM) and western-blotting. The ROS generation was measured by flow cytometry. RESULTS: Pure KillerRed was obtained with a yield of about 37 mg per liter of bacterial cells. KillerRed photodynamic inactivated the leukemia cells in a concentration-dependent manner, but exhibited no obvious dark toxicity. PDT mediated by KillerRed could also induce apoptotic response (mainly early apoptosis) in the three cell lines. The CLSM imaging indicated that KillerRed was distributed within the cytoplasm and nuclei of leukemia cells, causing damages to the cytoplasm and leaving the nuclear envelope intact during light irradiation. KillerRed distributed both in the cytosol and nuclei was confirmed by western blotting, and ROS significantly increased in PDT treated cells compared to the cells treated with KillerRed alone. CONCLUSIONS: Our studies demonstrated that KillerRed-mediated PDT could effectively inactivate K562, NB4, and THP1 leukemia cells and trigger cell apoptosis, and it has potential to be used individually or complementally, in the treatment of leukemia.

Protoberberine compounds extracted from Chelidonium majus L. as novel natural photosensitizers for cancer therapy.

BACKGROUND: It has been shown that secondary metabolites occur in Chelidonium majus L. (C. majus) crude extract and milky sap (alkaloids such as berberine, coptisine, chelidonine, chelerythrine, sanguinarine, and protopine) are biologically active compounds with a wide spectrum of pharmacological functions. Berberine, an isoquinoline alkaloid extracted from plants, possesses a wide range of biological activities, including inhibition of growth of a variety of cancer cell lines. PURPOSE AND STUDY DESIGN: In the present study, we investigated the potential anticancer effect of a protoberberine alkaloidal fraction (BBR-F) isolated from the medicinal plant C. majus on HeLa and C33A cervical cancer cells after light irradiation (PDT treatment). METHODS: BBR-F was prepared from an ethanolic extract of stems of C. majus. Identification of alkaloidal compounds was performed using high-performance liquid chromatography - mass spectrometry (HPLC/ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. BBR-F was then biologically evaluated for its anticancer properties. Cytotoxic activity after PDT treatment and without light irradiation (dark cytotoxicity) was determined by colorimetric WST-1 assay. The impact of the protoberberine alkaloidal fraction on the morphology and function of the cells was assessed by fluorescence and confocal microscopy as well as by flow cytometric analysis. To investigate the proinflammatory effect of the extracted natural BBR-F, nitric oxide concentration was determined using the Griess method. RESULTS: An effective reduction in HeLa and C33A cell viability was observed after PDT treatment of BBR-F treated cells. Furthermore, microscopic analysis identified various morphological changes in the studied cells that occurred during apoptosis. Apoptosis of HeLa and C33A cells was also characterized by biochemical changes in cell membrane composition, activation of intracellular caspases, disruption of the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) generation. CONCLUSION: Our results strongly suggest that the components of the natural plant protoberberine fraction (BBR-F) extracted from C. majus may represent promising novel photosensitive agents and can be applied in cancer photodynamic therapy as natural photosensitizers.

Photodynamic therapy with Bixa orellana extract and LED for the reduction of halitosis: study protocol for a randomized, microbiological and clinical trial.

BACKGROUND: Halitosis is an unpleasant breath odour that can interfere with the professional life, social life and quality of life of people who suffer from it. A modality of treatment that has been increasing in dentistry is antimicrobial photodynamic therapy (aPDT). Bixa orellana, popularly known as "urucum" is a plant native to Brazil. The seeds are used to produce a dye that is largely used in the food, textile, paint and cosmetic industries. The aim of this study is to verify whether aPDT with Bixa orellana extract and blue light-emitting diodes (LEDs) is effective in reducing halitosis. This method will also be compared with tongue scraping, the most commonly used conventional method for tongue coating removal, and the association of both methods will be evaluated. METHODS/DESIGN: A randomized clinical trial will be conducted at the dental clinic of the Universidade Nove de Julho. Thirty-nine patients will be divided by block randomization into three groups (n = 13) according to the treatment to be performed. In Group 1, tongue scraping will be performed by the same operator in all patients for analysis of the immediate results. Patients will also be instructed on how to use the scraper at home. Group 2 will be treated with aPDT with Bixa orellana extract and the LED light curing device: Valo Cordless Ultradent®. Six points in the tongue dorsum with a distance of 1 cm between them will be irradiated. The apparatus will be pre-calibrated at wavelength 395-480 nm for 20 s and 9.6 J per point. In Group 3, patients will be submitted to the tongue scraping procedure, as well as to the previously explained aPDT. Oral air collection with the Oral Chroma™ and microbiological collections of the tongue coating shall be done before, immediately after and 7 days after treatment for comparison. DISCUSSION: Halitosis treatment is a topic that still needs attention. The results of this trial could support decision-making by clinicians regarding aPDT using blue LEDs for treating halitosis on a daily basis, as most dentists already have this light source in their offices. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03346460 . Registered on 17 November 2017.

Photo-induced antibacterial activity of a porphyrin derivative isolated from the harmful dinoflagellate Heterocapsa circularisquama.

The dinoflagellate Heterocapsa circularisquama is highly toxic to bivalves. However, significant toxicity to finfish species has not been reported. We previously found that H. circularisquama has light-dependent haemolytic agents. Purification and chemical structural analyses revealed that the haemolytic agent H2-a is a porphyrin derivative, which exhibits light-dependent cytotoxicity toward tumour cells. To clarify the biological activity of H2-a further, its antibacterial activities against Gram-positive and Gram-negative bacteria were investigated in this study. A fraction (F5) equivalent to H2-a purified from the methanol extract of H. circularisquama showed potent light-dependent bactericidal activity toward Staphylococcus aureus, and the activity was concentration- and light illumination time-dependent; however, Escherichia coli was highly resistant to F5. Electron microscopic observation suggested that F5 induces morphological changes in S. aureus in a light-dependent manner. Further analysis using other bacterial species showed that the Gram-positive bacterium Bacillus subtilis was more sensitive than the Gram-negative bacteria Pseudomonas aeruginosa and Vibrio alginolyticus. These results indicate that F5 is a photo-induced antibacterial agent with relatively higher specificity to Gram-positive bacteria. Iodometric assay suggested that singlet oxygen was generated from light-illuminated F5. Histidine, a specific singlet oxygen scavenger, markedly inhibited the photosensitising antibacterial activity of F5 against S. aureus, suggesting the involvement of singlet oxygen in antibacterial activity. The antibacterial spectrum of F5 was evidently different from that of 5,10,15,20-tetra (N,N,N-trimethylanilinium) porphyrin tetratosylate, a commercially available porphyrin compound with antibacterial activity. Our results demonstrate that H. circularisquama has a novel antibacterial photosensitiser, a porphyrin derivative, with relatively higher specificity to Gram-positive bacteria. To the best of our knowledge, this is the first study to discover a porphyrin derivative with antibacterial activity in marine microalga.

Comparative study of phototoxicity of protoporphyrin IX synthetic and extracted from ssp Rattus novergicus albinus rats toward murine melanoma cells.

Protoporphyrin IX (PpIX) is a precursor of heme synthesis and is known to be an active photosensitizer and precursor of photosensitizers applied in photodynamic therapy (PDT) and photodynamic diagnostics (PDD). On irradiation with visible light, PpIX undergoes phototransformation, producing photoproducts which may also be phototoxic and increase its efficacy. The mechanism of PpIX phototransformation depends on environmental characteristics and can be different in vitro and in vivo. In this paper, we present a comparative study of the photoactivity of synthetic PpIX and PpIX extracted from the Harderian gland of ssp Rattus novergicus albinus rats, along with their photoproducts toward murine B16F-10 melanoma cells. It was observed that when irradiated with visible light the endogenous PpIX demonstrates photocytotoxicity ten times higher than the synthetic PpIX. The photoproduct of endogenous PpIX also possesses phototoxicity, though slightly lower than that of PpIX itself. The rate of cell internalization for both endogenous PpIX and its photoproduct was eightfold greater than that obtained for the synthetic porphyrin. This difference might result from a complexation of the native PpIX with some amphiphilic compounds during its synthesis within the Harderian glands, which facilitates the cell uptake of PpIX. Fluorescence microscopy images show that both endogenous and synthetic porphyrins are localized after uptake predominantly in the mitochondrial region of cells.

Quantification of a Novel Photosensitizer of Chlorin e6-C15-Monomethyl Ester in Beagle Dog Plasma Using HPLC: Application to Pharmacokinetic Studies.

Chlorin e6-C15-monomethyl ester (CMME) is a novel photosensitizer, which is synthetized from the degradation products of silkworm excrement. Preclinical studies on the promising photosensitizer CMME are necessary to determine its therapeutic efficacy and druglikeness. A high-performance liquid chromatography with UV detection (HPLC-UV) method was established for the determination of CMME in beagle dog plasma. The sample preparation involved a protein-precipitation method with acetonitrile after the addition of tanshinone IIA as an internal standard (IS). CMME and the IS were separated on a Diamonsil C18 (2) column (100 mm × 4.6 mm, 5 µm) with a isocratic system of methanol-water containing 20 mM ammonium acetate with 0.3% glacial acetic acid (85:15, v/v). The flow rate was 1.0 mL/min with UV detection using a wavelength of 400 nm. The method was sensitive enough with a lower limit of quantitation (LLOQ) of 0.05 µg/mL and had a good linearity (r² > 0.999) over the linear range of 0.05-5.00 µg/mL. The intra-day and inter-day accuracies ranged from 98.5% to 102.8% and precisions (RSD) were within 6.8%. The validated method was successfully applied to the pharmacokinetic study of CMME after intravenous administration of single and multiple doses in beagle dogs.

Photosensitizing anthraquinones from Heterophyllaea lycioides (Rubiaceae).

Seven anthraquinones were isolated from aerial parts of Heterophyllaea lycioides (Rusby) Sandwith (Rubiaceae), including three derivatives that have not been described before: a hetero-bianthraquinone identified as (R)-2-hydroxymethyl-2'methyl-1,1',6,6'-tetrahydroxy-5,5' bianthraquinone (lycionine), and two mono-chlorinated derivatives related to soranjidiol. One of them is a homo-bianthraquinone: (R)-7-chloro-2,2'-dimethyl-1,1',6,6'-tetrahydroxy-5,5' bianthraquinone (7-chlorobisoranjidiol), whereas the second halogenated derivative corresponds to a monomeric structure: 5-chloro-1,6-dihydroxy-2-methyl anthraquinone (5-chlorosoranjidiol). The four known compounds were already isolated from another species of this genus, H. pustulata, and they were identified as 5,5'-bisoranjidiol, soranjidiol, pustuline and heterophylline. Structural elucidation was performed by means of an extensive spectroscopic analysis, including 1D and 2D NMR data as well as by HRMS analysis. Chemical structures of 7-chlorobisoranjidiol and 5-chlorosoranjidiol were confirmed by their synthesis from 5,5'-bisoranjidiol and soranjidiol, respectively. Type I photosensitizing properties (superoxide anion radical generation, O2-) were assessed by using the nitroblue tetrazolium assay. When lycionine and chlorinated derivatives were irradiated, they enhanced the O2- production with respect to the control; 7-chlorobisoranjidiol stood out by generating an increase of 20%, whereas the other anthraquinones only produced a slight increase of 7%.

Preclinical Study of Antineoplastic Sinoporphyrin Sodium-PDT via In Vitro and In Vivo Models.

Photodynamic therapy (PDT) investigations have seen stable increases and the development of new photosensitizers is a heated topic. Sinoporphyrin sodium is a new photosensitizer isolated from Photofrin. This article evaluated its anticancer effects by clonogenic assays, MTT assays and xenograft experiments in comparison to Photofrin. The clonogenicity inhibition rates of sinoporphyrin sodium-PDT towards four human cancer cell lines ranged from 85.5% to 94.2% at 0.5 µg/mL under 630 nm irradiation of 30 mW/cm² for 180 s. For MTT assays, the IC50 ranges of Photofrin-PDT and sinoporphyrin sodium-PDT towards human cancer cells were 0.3 µg/mL to 5.5 µg/mL and 0.1 µg/mL to 0.8 µg/mL under the same irradiation conditions, respectively. The IC50 values of Photofrin-PDT and sinoporphyrin sodium-PDT towards human skin cells, HaCaT, were 10 µg/mL and 1.0 µg/mL, respectively. Esophagus carcinoma and hepatoma xenograft models were established to evaluate the in vivo antineoplastic efficacy. A control group, Photofrin-PDT group (20 mg/kg) and sinoporphyrin sodium group at three doses, 0.5 mg/kg, 1 mg/kg and 2 mg/kg, were set. Mice were injected with photosensitizers 24 h before 60 J 630 nm laser irradiation. The tumor weight inhibition ratio of 2 mg/kg sinoporphyrin sodium-PDT reached approximately 90%. Besides, the tumor growths were significantly slowed down by 2 mg/kg sinoporphyrin sodium-PDT, which was equivalent to 20 mg/kg Photofrin-PDT. In sum, sinoporphyrin sodium-PDT showed great anticancer efficacy and with a smaller dose compared with Photofrin. Further investigations are warranted.

Tectona grandis leaf extract, free and associated with nanoemulsions, as a possible photosensitizer of mouse melanoma B16 cell.

Over the past six years we have been studying extracts from tropical, specially Amazon, plants, to search for new sensitizers for photodynamic therapy of cancer and infectious diseases. Tectona grandis is a genus of tropical hardwood trees in the mint family, Lamiaceae. That is native to south and southeast Asia, but since the end of the 20th century is also gaining ground in the Amazon. The present work aims to evaluate the photodynamic potential of hydro-alcoholic extract from Tectona grandis LF leaves (TGE) and the same extract prepared as the oil-water nanoemulsion (TGE-NE) against melanoma B16 F10 cells. The method for preparation of a stable nanoemulsion with ~20nm particles associated to the TGE (TGE-NE) was successfully developed. We have shown that both free and nanostructured presentations possess the ability to sensitize B16 F10 cells to red light of the LED in vitro. Photodynamic effect was observed for both TGE and TGE-NE because toxicity increased under illumination with red light. While TGE was highly toxic towards melanoma cells under illumination with red light of the LED, it also possessed significant dark toxicity towards both B16 F10 and murine fibroblast NIH3T3 cells. The TGE-NE showed reasonable photocytotoxicity and was much less toxic towards normal cells in the dark compared to free TGE.

Parietin: an efficient photo-screening pigment in vivo with good photosensitizing and photodynamic antibacterial effects in vitro.

The photophysical, photoinduced pro-oxidant and antibacterial properties in vitro of the natural occurring parietin (PTN; 1,8-dihydroxy-3-methoxy-6-methyl-9,10-anthraquinone) were evaluated. PTN was extracted from the lichen identified as Teloschistes flavicans (Sw.) Norm. (Telochistaceae). Results indicate that in chloroform solution, PTN presents spectroscopic features corresponding to an excited-state intramolecular proton-transfer (ESIPT) state with partial keto-enol tautomerization. In argon-saturated solutions, the singlet excited state is poorly fluorescent (ΦF = 0.03), decaying by efficient intersystem crossing to an excited triplet state 3PTN*, as detected by laser-flash photolysis experiments. In the presence of triplet molecular oxygen, the 3PTN* was fully quenched producing singlet molecular oxygen (1O2) with a quantum yield of 0.69. In addition, in buffer solutions, PTN has the ability to also generate a superoxide radical anion (O2˙-) in a human leukocyte model and its production was enhanced under UVA-Vis irradiation. Finally, the in vitro antibacterial capability of PTN in the dark and under UVA-Vis illumination was compared in microbial cultures of both Gram positive and negative bacteria. As a result, PTN showed promising photo-induced antibacterial activity through the efficient photosensitized generation of both 1O2 and O2˙- species. Thus, we have demonstrated that PTN, an efficient photo-screening pigment in lichens, is also a good photosensitizer in solution with promising applications in antibacterial photodynamic therapy.

Inactivation of plant-pathogenic fungus Colletotrichum acutatum with natural plant-produced photosensitizers under solar radiation.

The increasing tolerance to currently used fungicides and the need for environmentally friendly antimicrobial approaches have stimulated the development of novel strategies to control plant-pathogenic fungi such as antimicrobial phototreatment (APT). We investigated the in vitro APT of the plant-pathogenic fungus Colletotrichum acutatum with furocoumarins and coumarins and solar radiation. The compounds used were: furocoumarins 8-methoxypsoralen (8-MOP) and 5,8-dimethoxypsoralen (isopimpinellin), coumarins 2H-chromen-2-one (coumarin), 7-hydroxycoumarin, 5,7-dimethoxycoumarin (citropten) and a mixture (3:1) of 7-methoxycoumarin and 5,7-dimethoxycoumarin. APT of conidia with crude extracts from 'Tahiti' acid lime, red and white grapefruit were also performed. Pure compounds were tested at 50µM concentration and mixtures and extracts at 12.5mgL(-1). The C. acutatum conidia suspension with or without the compounds was exposed to solar radiation for 1h. In addition, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP was the most effective treatment, killing 100% of the conidia followed by the mixture of two coumarins and isopimpinellin that killed 99% and 64% of the conidia, respectively. APT with the extracts killed from 20% to 70% of the conidia, and the extract from 'Tahiti' lime was the most effective. No damage to sweet orange leaves was observed after APT with any of the compounds or extracts.

Exerting better control and specificity with singlet oxygen experiments in live mammalian cells.

Singlet molecular oxygen, O2(a1Δg), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O2(a1Δg) in this regard is acknowledged, an understanding of the targets and mechanisms of O2(a1Δg) action is inadequate. Thus, methods that better facilitate studies of O2(a1Δg) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O2(a1Δg) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O2(a1Δg) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O2(a1Δg) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O2(a1Δg) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O2(a1Δg) at the expense of other ROS. We also comment on our recent efforts to monitor O2(a1Δg) in cells and to monitor the cell's response to O2(a1Δg).

Targeting T Cell Bioenergetics by Modulating P-Glycoprotein Selectively Depletes Alloreactive T Cells To Prevent Graft-versus-Host Disease.

T lymphocytes play a central role in many human immunologic disorders, including autoimmune and alloimmune diseases. In hematopoietic stem cell transplantation, acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Selectively depleting GVHD-causing cells prior to transplant may prevent GVHD. In this study, we evaluated 24 chalcogenorhodamine photosensitizers for their ability to selectively deplete reactive T lymphocytes and identified the photosensitizer 2-Se-Cl, which accumulates in stimulated T cells in proportion to oxidative phosphorylation. The photosensitizer is also a potent stimulator of P-glycoprotein (P-gp). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria and protects resting lymphocytes that are essential for antipathogen and antitumor responses. To evaluate the selective depletion of alloimmune responses, donor C57BL/6 splenocytes were cocultured for 5 d with irradiated BALB/c splenocytes and then photodepleted (PD). PD-treated splenocytes were infused into lethally irradiated BALB/c (same-party) or C3H/HeJ (third-party) mice. Same-party mice that received PD-treated splenocytes at the time of transplant lived 100 d without evidence of GVHD. In contrast, all mice that received untreated primed splenocytes and third-party mice that received PD-treated splenocytes died of lethal GVHD. To evaluate the preservation of antiviral immune responses, acute lymphocytic choriomeningitis virus infection was used. After photodepletion, expansion of Ag-specific naive CD8(+) T cells and viral clearance remained fully intact. The high selectivity of this novel photosensitizer may have broad applications and provide alternative treatment options for patients with T lymphocyte-mediated diseases.