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.

Synthesis, characterization, and biological evaluation of new Ru(II) polypyridyl photosensitizers for photodynamic therapy.

Two Ru(II) polypyridyl complexes, Ru(DIP)2(bdt) (1) and [Ru(dqpCO2Me)(ptpy)](2+) (2) (DIP = 4,7-diphenyl-1,10-phenanthroline, bdt = 1,2-benzenedithiolate, dqpCO2Me = 4-methylcarboxy-2,6-di(quinolin-8-yl)pyridine), ptpy = 4'-phenyl-2,2':6',2″-terpyridine) have been investigated as photosensitizers (PSs) for photodynamic therapy (PDT). In our experimental settings, the phototoxicity and phototoxic index (PI) of 2 (IC50(light): 25.3 µM, 420 nm, 6.95 J/cm(2); PI >4) and particularly of 1 (IC50(light): 0.62 µM, 420 nm, 6.95 J/cm(2); PI: 80) are considerably superior compared to the two clinically approved PSs porfimer sodium and 5-aminolevulinic acid. Cellular uptake and distribution of these complexes was investigated by confocal microscopy (1) and by inductively coupled plasma mass spectrometry (1 and 2). Their phototoxicity was also determined against the Gram-(+) Staphylococcus aureus and Gram-(-) Escherichia coli for potential antimicrobial PDT (aPDT) applications. Both complexes showed significant aPDT activity (420 nm, 8 J/cm(2)) against Gram-(+) (S. aureus; >6 log10 CFU reduction) and, for 2, also against Gram-(-) E. coli (>4 log10 CFU reduction).

Singlet oxygen generation in porphyrin-doped polymeric surface coating enables antimicrobial effects on Staphylococcus aureus.

Surfaces can be coated with photosensitizer molecules, which generate singlet oxygen ((1)O2) when the surface is exposed to light. (1)O2 may diffuse from the coating and has the potential to kill microorganisms present on the surface. In the present study a derivative of the meso-tetraphenylporphyrin (TPP) was immobilized onto polyurethane (PU) after being sprayed and polymerized as a thin layer onto poly-methylmethacrylate (PMMA). PU is gas permeable and thus a sufficient amount of oxygen reaches the photosensitizer in this coating. The surface generation of (1)O2 and its diffusion were investigated by detecting its luminescence at 1270 nm and a tri-iodide assay. Antimicrobial photodynamic surface effects were tested on Staphylococcus aureus. The spectrally resolved detection of (1)O2 luminescence yielded a clear peak at 1275 nm. The time-resolved luminescence showed multi-exponential decay, revealing rise and decay times in the range of 5-2 × 10(2)µs. The photodynamic inactivation of S. aureus was monitored at different photosensitizer concentrations and radiant exposures of light. A photodynamic killing of >99.9% (>3log10-steps) was achieved within an irradiation time of 30 min. The photodynamic killing on the bioactive surface confirmed the antimicrobial effect of (1)O2 that was generated in the PU-coating and reached the bacteria by diffusion.

Fungicidal photodynamic effect of a twofold positively charged porphyrin against Candida albicans planktonic cells and biofilms.

BACKGROUND: Antimicrobial photodynamic therapy is an interesting alternative for the treatment of superficial mucocutaneous mycoses. In immunodeficient patients, these infections are frequently recurrent and resistant to the most commonly used antifungal medications. Candida albicans biofilms frequently cause such infections that can even evolve to deep-seated mycoses. MATERIALS & METHODS: The efficiency of a photodynamic therapy was investigated against C. albicans using a twofold positively charged porphyrin (XF-73) in comparison with the well-known fourfold positively charged porphyrin (5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate salt). RESULTS: After incubation with 0.5 µM of XF-73 for 15 min and irradiation with blue light (12.1 J/cm(2)), the viability of C. albicans planktonic cells decreased by over 6 log10. For biofilm cells, a longer incubation time (4 h) with 1 µM of XF-73 and a light dose of 48.2 J/cm(2) was necessary to achieve over 5 log10 cell killing. Cell killing was mediated by singlet oxygen that was directly detected via its luminescence at 1270 nm in XF-73-incubated C. albicans biofilms for the first time. Antimicrobial photodynamic therapy yielded better results for XF-73 compared with 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate salt when using the same conditions. CONCLUSION: This study provides evidence that XF-73 is a highly efficient photosensitizer to kill C. albicans and it would be worthwhile to test this photosensitizer in clinical studies for both prophylaxis and treatment of infections caused by this microorganism, preventing the spread of C. albicans throughout the bloodstream.

Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in Candida albicans cells.

Singlet oxygen (¹O2) is an important reactive intermediate in photodynamic reactions, particularly in antimicrobial PDT (aPDT). The detection of ¹O2 luminescence is frequently used to elucidate the role of ¹O2 in various environments, particularly in microorganisms and human cells. When incubating the fungus, Candida albicans, with porphyrins XF73 (5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin) or TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate)), the ¹O2 luminescence signals were excellent for TMPyP. In case of XF73, the signals showed strange rise and decay times. Thus, ¹O2 generation of XF73 was investigated and compared with TMPyP. Absorption spectroscopy of XF73 showed a change in absorption cross section when there was a change in the concentration from 1×10⁻6M to 1×10⁻³ M indicating an aggregation process. The addition of phosphate buffered saline (PBS) substantially changed ¹O2 luminescence in XF73 solution. Detailed experiments provided evidence that the PBS constituents NaCl and KCl caused the change of ¹O2 luminescence. The results also indicate that Cl- ions may cause aggregation of XF73 molecules, which in turn enhances self-quenching of ¹O2 via photosensitizer molecules. These results show that some ions, e.g., those present in cells in vitro or added by PBS, can considerably affect the detection and the interpretation of time-resolved luminescence signals of ¹O2, particularly in in vitro and in vivo. These effects should be considered for any other photosensitizer used in photodynamic processes.

Hydrogen bond acceptors and additional cationic charges in methylene blue derivatives: photophysics and antimicrobial efficiency.

Photodynamic inactivation of bacteria (PIB) by efficient singlet oxygen photosensitizers might be a beneficial alternative to antibiotics in the struggle against multiresistant bacteria. Phenothiazinium dyes belong to the most prominent classes of such sensitizers due to their intense absorption in the red-light region (λ(abs, max) ca. 600-680 nm, ε > 50,000 L mol(-1) cm(-1)), their low toxicity, and their attachment/penetration abilities. Except simple substituents like alkyl or hydroxyalkyl residues, nearly no modifications of the phenothiaziniums have been pursued at the auxochromic sites. By this, the properties of methylene blue derivatives and their fields of application are limited; it remains unclear if their potential antimicrobial efficacy may be enhanced, also to compete with porphyrins. We prepared a set of six mainly novel methylene blue derivatives with the ability of additional hydrogen bonding and/or additional cationic charges to study the substituents' effect on their activity/toxicity profiles and photophysical properties. Direct detection of singlet oxygen was performed at 1270 nm and the singlet oxygen quantum yields were determined. In suspensions with both, gram-positive and gram-negative bacteria, some derivatives were highly active upon illumination to inactivate S. aureus and E. coli up to 7 log10 steps (99.99999%) without inherent toxicities in the nonirradiated state.

Dirty hands: photodynamic killing of human pathogens like EHEC, MRSA and Candida within seconds.

Hand hygiene is one of the most important interventions for reducing transmission of nosocomial life-threatening microorganisms, like methicillin resistant Staphylococcus aureus (MRSA), enterohemorrhagic Escherichia coli (EHEC) or Candida albicans. All three pathogens have become a leading cause of infections in hospitals. Especially EHEC is causing severe diarrhoea and, in a small percentage of cases, haemolytic-uremic syndrome (HUS) as reported for E. coli 104:H4 in Germany 2011. We revealed the possibility to inactivate very fast and efficiently MRSA, EHEC and C. albicans using the photodynamic approach. MRSA, EHEC and C. albicans were incubated in vitro with different concentrations of TMPyP for 10 s and illuminated with visible light (50 mW cm(-2)) for 10 and 60 s. 1 µmol l(-1) of TMPyP and an applied radiant exposure of 0.5 J cm(-2) achieved a photodynamic killing of ≥99.9% of MRSA and EHEC. Incubation with higher concentrations (up to 100 µmol l(-1)) of TMPyP caused bacteria killing of >5 log(10) (≥99.999%) after illumination. Efficient Candida killing (≥99.999%) was achieved first at a higher light dose of 12 J cm(-2). Different rise and decay times of singlet oxygen luminescence signals could be detected in Candida cell suspensions for the first time, indicating different oxygen concentrations in the surrounding for the photosensitizer and singlet oxygen, respectively. This confirms that TMPyP is not only found in the water-dominated cell surrounding, but also within the C. albicans cells. Applying a water-ethanol solution of TMPyP on ex vivo porcine skin, fluorescence microscopy of histology showed that the photosensitizer was exclusively localized in the stratum corneum regardless of the incubation time. TMPyP exhibited a fast and very effective killing rate of life-threatening pathogens within a couple of seconds that encourages further testing in an in vivo setting. Being fast and effective, antimicrobial photodynamic applications might become acceptable as a tool for hand hygiene procedures and also in other skin areas.

Fast and effective: intense pulse light photodynamic inactivation of bacteria.

The goal of this study was to investigate the photodynamic toxicity of TMPyP (5, 10, 15, 20-Tetrakis (1-methylpyridinium-4-yl)-porphyrin tetra p-toluenesulfonate) in combination with short pulses (ms) of an intense pulse light source within 10 s against Bacillus atrophaeus, Staphylococcus aureus, Methicillin-resistant S. aureus and Escherichia coli, major pathogens in food industry and in health care, respectively. Bacteria were incubated with a photoactive dye (TMPyP) that is subsequently irradiated with visible light flashes of 100 ms to induce oxidative damage immediately by generation of reactive oxygen species like singlet oxygen. A photodynamic killing efficacy of up to 6 log(10) (>99.9999%) was achieved within a total treatment time of 10 s using a concentration range of 1-100 µmol TMPyP and multiple light flashes of 100 ms (from 20 J cm(-2) up to 80 J cm(-2)). Both incubation of bacteria with TMPyP alone or application of light flashes only did not have any negative effect on bacteria survival. Here we could demonstrate for the first time that the combination of TMPyP as the respective photosensitizer and a light flash of 100 ms of an intense pulsed light source is enough to generate sufficient amounts of reactive oxygen species to kill these pathogens within a few seconds. Increasing antibiotic resistance requires fast and efficient new approaches to kill bacteria, therefore the photodynamic process seems to be a promising tool for disinfection of horizontal surfaces in industry and clinical purposes where savings in time is a critical point to achieve efficient inactivation of microorganisms.

UVA and endogenous photosensitizers--the detection of singlet oxygen by its luminescence.

UVA irradiation (320-400 nm) comprises about 95 percent of incident midday solar ultraviolet irradiation. It penetrates skin much deeper than UVB irradiation. The absorption of UVA irradiation in endogenous chromophores frequently leads to the generation of reactive oxygen species such as singlet oxygen ((1)O(2)). (1)O(2) is an important biochemical intermediate in multiple biological processes. Beside other procedures, the direct detection of (1)O(2) by its luminescence is a powerful tool that helps to understand the generation of (1)O(2) during UVA exposure in solution, in vitro and in vivo. This article describes the endogenous photosensitizers, their ability to generate (1)O(2) under UVA irradiation, and the detection technology to visualize the action of (1)O(2).

Photodynamic inactivation of multi-resistant bacteria (PIB) - a new approach to treat superficial infections in the 21st century.

The increasing resistance of bacteria against antibiotics is one of the most important clinical challenges of the 21(st) century. Within the gram-positive bacteria the methicillin-resistant Staphylococcus aureus and Enterococcus faecium represent the major obstacle to successful therapy. Apart from the development of new antibiotics it requires additional differently constituted approaches, like photodynamic inactivation in order to have further effective treatment options against bacteria available. Certain dyes, termed photosensitizers, are able to store the absorbed energy in long-lived electronic states upon light activation with appropriate wavelengths and thus make these states available for chemical activation of the immediate surroundings. The interaction with molecular oxygen, which leads to different, very reactive and thus cytotoxic oxygen species, is highlighted. In this review the application of the photodynamic inactivation of bacteria will be discussed regarding the possible indications in dermatology, like localized skin and wound infections or the reduction of nosocomial colonization with multi-resistant bacteria on the skin. The crucial advantage of the local application of photosensitizers followed by irradiation of the area of interest is the fact that independent of the resistance pattern of a bacterium a direct inactivation takes place similarly as with an antiseptic. In this review the physical-chemical and biological basics of photo-dynamic inactivation of bacteria (PIB) will be discussed as well as the possible dermatological indications.

A helpful technology--the luminescence detection of singlet oxygen to investigate photodynamic inactivation of bacteria (PDIB).

Photodynamic inactivation of bacteria (PDIB) is considered a new approach for the struggle against multiresistant bacteria. To achieve a sufficient level of bacteria killing, the photosensitizer must attach to and/or penetrate the bacteria and generate a sufficiently high amount of singlet oxygen. To optimize PDIB, the direct detection and quantification of singlet oxygen in bacteria is a helpful tool. Singlet-oxygen luminescence is a very weak signal, in particular in living bacteria. We first performed experiments in aqueous photosensitizer solution to optimize the luminescence system. We eliminated non-singlet-oxygen photons, which is important for the quantification of singlet oxygen and its rise and decay rates. This procedure is even more important when the laser excitation beam is scattered by bacteria (diameter 1 microm). In suspensions with both Gram-positive and Gram-negative bacteria we then clearly detected singlet oxygen by its luminescence and determined the respective rise and decay times. The decay times should provide an indication of localization of singlet oxygen and hence of the photosensitizer even in small bacteria.