A novel set of symmetric methylene blue derivatives exhibits effective bacteria photokilling - a structure-response study.

This study focuses on the structure-response relationship of symmetrically substituted phenothiazinium dyes. Four hydrophilic derivatives with the ability of additional hydrogen bonding (, ) or additional electrostatic interaction (, ) were synthesized, photophysically characterized and compared to the parent compound methylene blue (MB, ) and a lipophilic derivative () without additional coordination sites. Derivative was most effective against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli reaching a maximum photodynamic efficacy of >5log10 steps (≥99.999%) of bacteria killing in 10 minutes (5 µM, 30 J cm(-2)) without inherent dark toxicity after one single treatment with the incoherent light source PDT1200 (λmax = 660 nm, 50 mW cm(-2)). Interestingly, one derivative with two additional primary positive charges () showed selective killing of Escherichia coli (5 µM, 30 J cm(-2), 4log10 steps inactivation (≥99.99%)) and no antimicrobial effect on Staphylococcus aureus. This might allow the development of a new generation of photosensitizers with higher antimicrobial efficacy and selectivity for future applications.

Fast and effective inactivation of Bacillus atrophaeus endospores using light-activated derivatives of vitamin B2.

Highly resistant endospores may cause severe problems in medicine as well as in the food and packaging industries. We found that bacterial endospores can be inactivated quickly with reactive oxygen species (ROS) that were generated by a new generation of flavin photosensitizers. Flavins like the natural compound vitamin B2 are already known to produce ROS but they show a poor antimicrobial photodynamic killing efficacy due to the lack of positive charges. Therefore we synthesized new flavin photosensitizers that have one (FLASH-01a) or eight (FLASH-07a) positive charges and can hence attach to the negatively charged surface of endospores. In this study we used standardized Bacillus atrophaeus endospores (ATCC 9372) as a biological surrogate model for a proof-of-concept study of photodynamic inactivation experiments using FLASH-01a and FLASH-07a. After incubation of spores with different flavin concentrations, the flavin derivatives were excited with blue light at a light dose of 70 J cm(-2). The inactivation of spores was investigated either in suspension or after attachment to polyethylene terephthalate (PET) surfaces. Incubation of spores suspended in Millipore water with 4 mM FLASH-01a for 10 seconds and irradiation with blue light for 10 seconds caused a biologically relevant decrease of spore survival of 3.5 log10 orders. Using FLASH-07a under the same conditions we achieved a decrease of 4.4 log10 orders. Immobilized spores on PET surfaces were efficiently killed with 7.0 log10 orders using 8 mM FLASH-07a. The total treatment time (incubation + irradiation) was as short as 20 seconds. The results of this study show evidence that endospores can be fastly and effectively inactivated with new generations of flavin photosensitizers that may be useful for industrial or medical applications in the future.

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.

Aarhus sensor green: a fluorescent probe for singlet oxygen.

A tetrafluoro-substituted fluorescein derivative covalently linked to a 9,10-diphenyl anthracene moiety has been synthesized, and its photophysical properties have been characterized. This compound, denoted Aarhus Sensor Green (ASG), has distinct advantages for use as a fluorescent probe for singlet molecular oxygen, O2(a(1)Δg). In the least, ASG overcomes several limitations inherent to the use of the related commercially available product called Singlet Oxygen Sensor Green (SOSG). The functional behavior of both ASG and SOSG derives from the fact that these weakly fluorescent compounds rapidly react with singlet oxygen via a π2 + π4 cycloaddition to irreversibly yield a highly fluorescent endoperoxide. The principal advantage of ASG over SOSG is that, at physiological pH values, both ASG and the ASG endoperoxide (ASG-EP) do not themselves photosensitize the production of singlet oxygen. As such, ASG better fits the requirement of being a benign probe. Although ASG readily enters a mammalian cell (i.e., HeLa) and responds to the presence of intracellular singlet oxygen, its behavior in this arguably complicated environment requires further investigation.

Blue light kills Aggregatibacter actinomycetemcomitans due to its endogenous photosensitizers.

OBJECTIVES: The aim of this study was to demonstrate that the periodontal pathogen Aggregatibacter actinomycetemcomitans (AA) can be killed by irradiation with blue light derived from a LED light-curing unit due to its endogenous photosensitizers. MATERIALS AND METHODS: Planktonic cultures of AA and Escherichia coli were irradiated with blue light from a bluephase® C8 light-curing unit with an emission peak at 460 nm, which is usually applied for polymerization of dental resins. A CFU-assay was performed for the analysis of viable bacteria after treatment. Moreover, bacterial cells were lysed and the lysed AA and E. coli were investigated for generation of singlet oxygen. Spectroscopic measurements of lysed AA and E. coli were performed and analyzed for characteristic absorption and emission peaks. RESULTS: A light dose of 150 J/cm(2) induced a reduction of ≥5 log10 steps of viable AA, whereas no effect of blue light was found against E. coli. Spectrally resolved measurements of singlet oxygen luminescence showed clearly that a singlet oxygen signal is generated from lysed AA upon excitation at 460 nm. Spectroscopic measurements of lysed AA exhibited characteristic absorption and emission peaks similar to those of known porphyrins and flavins. CONCLUSIONS: AA can be inactivated by irradiation with blue light only, without application of an exogenous photosensitizer. CLINICAL RELEVANCE: These results encourage further studies on the potential use of these blue light-mediated auto-photosensitization processes in the treatment of periodontitis for the successful inactivation of Aggregatibacter actinomycetemcomitans.

Luminescence spectroscopy of singlet oxygen enables monitoring of oxygen consumption in biological systems consisting of fatty acids.

The interaction of singlet oxygen ((1)O2) generated in a photosensitized process with well-known reference photosensitizers Perinaphthenone (PN) and TMPyP is investigated in a model system consisting of fatty acids and the respective exogenous photosensitizer (PS) in solution by direct detection of the luminescence photons of (1)O2 at 1270 nm. Such a model system is a first approach to mimic the complex environment of (1)O2 in a biological cell which consists mainly of water, proteins, sugars and lipids. Firstly, the important issue of oxygen consumption is evaluated which has to be considered during luminescence detection of (1)O2. It is known that the luminescence signal of (1)O2 is dependent on the oxygen concentration of the environment. Cellular components such as lipids represent oxygen consumers due to peroxidation of their unsaturated double bonds. Secondly, the experimental conditions for this model system regarding oxygen consumption are optimized to estimate the rates and rate constants of the coupled system. Thirdly, the triplet decay of the PS can provide more precise information about the actual oxygen concentration close to the PS and can be used, therefore, as a more precise method to determine the oxygen concentration in more complex systems such as a biological cell. The aim is to get a better understanding of photosensitized reactions of (1)O2 with cellular components to further improve methodologies, in particular at a cellular level using luminescence spectroscopy. In conclusion, luminescence detection might be a helpful tool to monitor precisely and promptly changes in oxygen concentration in a complex environment.

UVA irradiation of fatty acids and their oxidized products substantially increases their ability to generate singlet oxygen.

UVA radiation plays an important role for adverse reactions in human tissue. UVA penetrates epidermis and dermis of skin being absorbed by various biomolecules, especially endogenous photosensitizers. This may generate deleterious singlet oxygen ((1)O2) that oxidizes fatty acids in cell membranes, lipoproteins, and other lipid-containing structures such as the epidermal barrier. Indications exist that fatty acids are not only the target of (1)O2 but also act as potential photosensitizers under UVA irradiation, if already oxidized. Five different fatty acids in ethanol solution (stearic, oleic, linoleic, linolenic and arachidonic acid) were exposed to UVA radiation (355 nm, 100 mW) for 30 seconds. (1)O2 luminescence was detected time-resolved at 1270 nm and confirmed in spectrally-resolved experiments. The more double bonds fatty acids have the more (1)O2 photons were detected. In addition, fatty acids were continuously exposed to broadband UVA for up to 240 min. During that time span, UVA absorption and (1)O2 luminescence substantially increased with irradiation time, reached a maximum and decreased again. HPLC-MS analysis showed that the amount of peroxidized fatty acids and the (1)O2 generation increased and decreased in parallel. This indicates the high potential of peroxidized fatty acids to produce (1)O2 under UVA irradiation. In conclusion, fatty acids along with peroxidized products are weak endogenous photosensitizers but become strong photosensitizers under continuous UVA irradiation. Since fatty acids and their oxidized products are ubiquitous in living cells and in skin, which is frequently and long-lasting exposed to UVA radiation, this photosensitizing effect may contribute to initiation of deleterious photooxidative processes in tissue.

Spatial Distribution of a Porphyrin-Based Photosensitizer Reveals Mechanism of Photodynamic Inactivation of Candida albicans.

The antimicrobial photodynamic therapy (aPDT) is a promising approach for the control of microbial and especially fungal infections such as mucosal mycosis. TMPyP [5,10,15, 20-tetrakis(1-methylpyridinium-4-yl)-porphyrin tetra p-toluenesulfonate] is an effective photosensitizer (PS) that is commonly used in aPDT. The aim of this study was to examine the localization of TMPyP in Candida albicans before and after irradiation with visible light to get information about the cellular mechanism of antifungal action of the photodynamic process using this PS. Immediately after incubation of C. albicans with TMPyP, fluorescence microscopy revealed an accumulation of the PS in the cell envelope. After irradiation with blue light the complete cell showed red fluorescence, which indicates, that aPDT is leading to a damage in the cell wall with following influx of PS into the cytosol. Incubation of C. albicans with Wheat Germ Agglutinin (WGA) could confirm the cell wall as primary binding site of TMPyP. The finding that the porphyrin accumulates in the fungal cell wall and does not enter the interior of the cell before irradiation makes it unlikely that resistances can emerge upon aPDT. The results of this study may help in further development and modification of PS in order to increase efficacy against fungal infections such as those caused by C. albicans.

Tattoo inks contain polycyclic aromatic hydrocarbons that additionally generate deleterious singlet oxygen.

In the past years, tattoos have become very popular worldwide, and millions of people have tattoos with mainly black colours. Black tattoo inks are usually based on soot, are not regulated and may contain hazardous polycyclic aromatic hydrocarbons (PAHs). Part of PAHs possibly stay lifelong in skin, absorb UV radiation and generate singlet oxygen, which may affect skin integrity. Therefore, we analysed 19 commercially available tattoo inks using HPLC and mass spectrometry. The total concentrations of PAHs in the different inks ranged from 0.14 to 201 microg/g tattoo ink. Benz(a)pyrene was found in four ink samples at a mean concentration of 0.3 +/- 0.2 microg/g. We also found high concentrations of phenol ranging from 0.2 to 385 microg/g tattoo ink. PAHs partly show high quantum yields of singlet oxygen (Phi(Delta)) in the range from 0.18 to 0.85. We incubated keratinocytes with extracts of different inks. Subsequent UVA irradiation decreased the mitochondrial activity of cells when the extracts contained PAHs, which sufficiently absorb UVA and show simultaneously high Phi(Delta) value. Tattooing with black inks entails an injection of substantial amounts of phenol and PAHs into skin. Most of these PAHs are carcinogenic and may additionally generate deleterious singlet oxygen inside the dermis when skin is exposed to UVA (e.g. solar radiation).

Real-time imaging of photodynamic action in bacteria.

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly-sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single-cell level. Imaging experiments in deuterated buffer strongly support a Type-I mechanism for methylene blue and a very minor role of singlet oxygen.

Fast and effective photodynamic inactivation of multiresistant bacteria by cationic riboflavin derivatives.

Photodynamic inactivation of bacteria (PIB) proves to be an additional method to kill pathogenic bacteria. PIB requires photosensitizer molecules that effectively generate reactive oxygen species like singlet oxygen when exposed to visible light. To allow a broad application in medicine, photosensitizers should be safe when applied in humans. Substances like vitamin B2, which are most likely safe, are known to produce singlet oxygen upon irradiation. In the present study, we added positive charges to flavin derivatives to enable attachment of these molecules to the negatively charged surface of bacteria. Two of the synthesized flavin derivatives showed a high quantum yield of singlet oxygen of approximately 75%. Multidrug resistant bacteria like MRSA (Methicillin resistant Staphylococcus aureus), EHEC (enterohemorrhagic Escherichia coli), Pseudomonas aeruginosa, and Acinetobacter baumannii were incubated with these flavin derivatives in vitro and were subsequently irradiated with visible light for seconds only. Singlet oxygen production in bacteria was proved by detecting its luminescence at 1270 nm. After irradiation, the number of viable bacteria decreased up to 6 log10 steps depending on the concentration of the flavin derivatives and the light dosimetry. The bactericidal effect of PIB was independent of the bacterial type and the corresponding antibiotic resistance pattern. In contrast, the photosensitizer concentration and light parameters used for bacteria killing did not affect cell viability of human keratinocytes (therapeutic window). Multiresistant bacteria can be safely and effectively killed by a combination of modified vitamin B2 molecules, oxygen and visible light, whereas normal skin cells survive. Further work will include these new photosensitizers for topical application to decolonize bacteria from skin and mucosa.

A comprehensive tutorial on in vitro characterization of new photosensitizers for photodynamic antitumor therapy and photodynamic inactivation of microorganisms.

In vitro research performed on eukaryotic or prokaryotic cell cultures usually represents the initial step for characterization of a novel photosensitizer (PS) intended for application in photodynamic therapy (PDT) of cancer or photodynamic inactivation (PDI) of microorganisms. Although many experimental steps of PS testing make use of the wide spectrum of methods readily employed in cell biology, special aspects of working with photoactive substances, such as the autofluorescence of the PS molecule or the requirement of light protection, need to be considered when performing in vitro experiments in PDT/PDI. This tutorial represents a comprehensive collection of operative instructions, by which, based on photochemical and photophysical properties of a PS, its uptake into cells, the intracellular localization and photodynamic action in both tumor cells and microorganisms novel photoactive molecules may be characterized for their suitability for PDT/PDI. Furthermore, it shall stimulate the efforts to expand the convincing benefits of photodynamic therapy and photodynamic inactivation within both established and new fields of applications and motivate scientists of all disciplines to get involved in photodynamic research.

Photodynamic biofilm inactivation by SAPYR--an exclusive singlet oxygen photosensitizer.

Prevention and control of biofilm-growing microorganisms are serious problems in public health due to increasing resistances of some pathogens against antimicrobial drugs and the potential of these microorganisms to cause severe infections in patients. Therefore, alternative approaches that are capable of killing pathogens are needed to supplement standard treatment modalities. One alternative is the photodynamic inactivation of bacteria (PIB). The lethal effect of PIB is based on the principle that visible light activates a photosensitizer, leading to the formation of reactive oxygen species, e.g., singlet oxygen, which induces phototoxicity immediately during illumination. SAPYR is a new generation of photosensitizers. Based on a 7-perinaphthenone structure, it shows a singlet oxygen quantum yield ΦΔ of 99% and is water soluble and photostable. Moreover, it contains a positive charge for good adherence to cell walls of pathogens. In this study, the PIB properties of SAPYR were investigated against monospecies and polyspecies biofilms formed in vitro by oral key pathogens. SAPYR showed a dual mechanism of action against biofilms: (I) it disrupts the structure of the biofilm even without illumination; (II) when irradiated, it inactivates bacteria in a polymicrobial biofilm after one single treatment with an efficacy of ≥ 99.99%. These results encourage further investigation on the potential of PIB using SAPYR for the treatment of localized infectious diseases.

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.