Partition of rose bengal anion from aqueous medium into a lipophilic environment in the cell envelope of Salmonella typhimurium: implications for cell-type targeting in photodynamic therapy

Photodynamic therapy employs photosensitizers for the selective destruction of tumor tissue while sparing the surrounding healthy tissue. Photosensitization may also be applied to the selective eradication of microorganisms. Photosensitized inactivation requires that the sensitizer bind to the target and therefore the factors that determine photosensitizer binding are critical to photosensitization selectivity. This paper reports the determination of some features of the binding site of the potent photosensitizer, Rose Bengal, in Salmonella bacteria and describes some of the factors that affect this binding. The shift in the wavelength of maximum fluorescence and experiments with the fluorescence quencher TNBS indicate that Rose Bengal is located in a non-aqueous compartment such as the outer membrane. The dye does not seem to significantly accumulate inside the cell, but rather to accumulate in the outer membrane. Time-dependent changes in sensitizer localization in two strains of Salmonella typhimurium that differ in cell wall formation, LT-2 and TA1975, correspond to their differences in susceptibility to photosensitized killing. Therefore these results provide clues to the factors that determine photosensitization selectivity. Understanding this phenomenon is essential for the efficient design of selective photosensitizers and for optimizing antitumor and antiviral photodynamic therapy.     

PHOTOINACTIVATION OF INFLUENZA VIRUS FUSION AND INFECTIVITY BY ROSE BENGAL

Rose bengal inactivated influenza virus upon exposure to light. Infectivity and fusion were inactivated with the same dose dependence, supporting the suggestion that the virucidal activity of photodynamic agents against enveloped viruses may be generally due to inactivation of their fusion protein(s). Concentrations required for inac-ti vation were found to depend upon the ratio of rose bengal to virus, rather than on the nominal aqueous concentration. Fusion-competent virosomes were inactivated similarly to intact virus particles. The HA_Z portion of the influenza fusion protein HA underwent two different, apparently mutually exclusive modifications upon illumination with rose bengal: cross-linking, and conversion to a form that moved slightly more slowly on sodium dodecyl sulfate poly-acrylamide gel electrophoresis. Inactivation of viral fusion was inhibited by oxygen removal or addition of azide or β-carotene, and was enhanced by D₂O, consistent with partial involvement of singlet oxygen. The possibility of a second mechanism of viral photoinactivation, by direct interaction between the viral fusion protein and the pho-toactivated dye, is also discussed.     

Calcium as a modulator of photosensitized killing of H9c2 cardiac cells.

Illumination of H9c2 rat heart cells in the presence of Rose Bengal resulted in dose-dependent cell killing (assessed by trypan blue staining) and modification of ionic currents flowing through the heart cell membrane. Inhibitors of voltage-gated ionic currents were shown to have little effect on cell killing. Ionic current measurements were used to assess the increase in leak conductance of these cells, which has been suggested to be a causal factor in killing of other cell types (1). Inhibitors of voltage-gated ionic currents, including the sodium channel blocker tetrodotoxin (100 microM) and the calcium channel blocker lanthanum (10 microM) were shown to have little effect on cell killing. The potassium channel inhibitor tetraethylammonium (20 mM) inhibited cell killing, but the effect is viewed as being caused by an inhibition of leak current. The time course of block of voltage-activated ionic currents during illumination, in the presence of Rose Bengal, was rapid compared with that for induction of leak current and for cell killing. These observations are consistent with a role for leak current in photosensitized killing of cardiac cells. They are interpreted with respect to calcium influx through the leak current pathway as a trigger for the cellular response.     

LETHAL EFFECTS OF NATURAL SOLAR ULTRAVIOLET RADIATION IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI: ACTIONS AND INTERACTIONS

Abstract— The inactivation of repair proficient ( Escherichia coli K12 AB 1157, E. coli B/r) and repair deficient ( E. coli K12 AB 1886 uvrA , AB 2463 recA and AB 2480 uvrA recA ) strains of bacteria by noon sunlight has been measured. The use of biological dosimetry based on an ultraviolet (UV) sensitive strain of Bacillus subtilis spores has allowed a quantitative comparison of bacterial inactivation by solar, 254 and 302 nm radiations. Our analysis indicates that: (1) uvrA and recA gene products are involved in repair of a substantial portion of the solar DNA damage, (2) 302 nm is a more appropriate wavelength than 254 nm to represent the DNA-damaging action of sunlight and that (3) repair proficient strains are inactivated by sunlight more rapidly than expected from the levels of DNA damage induced. When populations of repair proficient bacteria are exposed to noon sunlight for 20 min, they become sensitive to the lethal action of far-UV (254 nm), MMS (0.1 M ) and to a lesser extent, mild heat (52°C).     

Photodynamic effects of hematoporphyrin-derivative on enzyme activities of murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative resulted in the inactivation of cytosolic, mitochondrial and lysosomal enzymes and in a decrease in cellular adenosine triphosphate and reduced glutathione concentrations. Comparison of these results with those of previous studies revealed that transmembrane transport systems and DNA repair enzymes are inactivated after much shorter illumination periods than are intracellular enzymes. Although the pattern of photodynamic damage altered by varying the protocol of preincubation with hematoporphyrin-derivative and washing, it appeared that under all experimental conditions the plasma membrane was much more sensitive to photodynamic damage than were the intracellular enzymes. Lysosomal membrane disruption with subsequent detrimental release of lysosomal enzymes has been implicated previously in certain forms of porphyrin-induced photodynamic cell destruction. Cytochemical studies on enzyme localization virtually exclude such a mechanism in hematoporphyrin-derivative-induced cell inactivation in L929 fibroblasts.     

PHOTODYNAMIC INACTIVATION OF AN ION CHANNEL: GRAMICIDIN A

The ion channel formed by the peptide gramicidin A in planar lipid membranes is inactivated by visible light in the presence of the photosensitizer Rose Bengal. This is concluded from the strong decrease of the membrane conductance by more than two orders of magnitude. Experiments performed at different oxygen concentrations, in the presence of the singlet oxygen quenchers beta-carotene or alpha-tocopherol indicate, that presumably a type I process between the dye Rose Bengal and the tryptophan residues of the gramicidin channel with a subsequent oxidation of the tryptophans is responsible for the loss of the conductance properties of the channel.     

Highly efficient and photostable photosensitizer based on BODIPY chromophore

Photosensitizers are reagents that produce reactive oxygen species upon light illumination and are commonly used to study oxidative stress or for photodynamic therapy. There are many available photosensitizers, but most have limitations, such as low photostability, structural instability, or a limited usable range of solvent conditions. Here, we describe a novel photosensitizer scaffold (2I-BDP) based on the unique characteristics of the BODIPY chromophore (i.e., high extinction coefficient, high photostability, and insensitivity to solvent environment). 2I-BDP shows stronger near-infrared singlet oxygen luminescence emission and higher photostability than the well-known photosensitizer, Rose Bengal. Unlike other photosensitizers, this scaffold is widely applicable under various conditions, including lipophilic and aqueous environments. HeLa cells loaded with 2I-BDP could be photosensitized by light illumination, demonstrating that 2I-BDP is potentially useful as a reagent for cell photosensitization, oxidative stress studies, or PDT.     

ENHANCING EFFECT OF ASCORBATE ON TOLUIDINE BLUE-PHOTOSENSITIZATION OF YEAST CELLS

Abstract— Using toluidine blue, a potent photosensitizer with a ₁O₂ dominated mechanism in yeast cell inactivation, it was found that addition of ascorbate to the sensitizer-cell mixture during illumination enhanced the inactivation. The enhancement required the presence of oxygen in the reaction mixture. The same enhancement was observed with methylene blue and thionine but not with xanthenes (Rose Bengal and eosin Y). The consumption of O₂ and ascorbate seemed coupled in the enhancement. From the observation that the presence of ascorbate for a very short time (1 s) in the reaction mixture was enough to exhibit the same enhancement, it was concluded that the ascorbate enhancement processes are probably initiated in bulk medium, not intracellularly. The ascorbate enhancement may be a combined consequence of the high electron-accepting property of triplet toluidine blue and the strong tendency of ascorbate to act as an electron donor. The role of oxygen was not specified whether it was directly involved in the photoinactivation of cells. Addition of N J appeared to suppress the photoinacti-vation only in the higher fluence region where ascorbate had been consumed. Thus the ascorbate enhancement seems to occur under low fluence conditions and may probably be independent of the singlet oxygen mechanism.     

Eradication of Gram‐Positive and Gram‐Negative Bacteria by Photosensitizers Immobilized in Polystyrene

Immobilization of photosensitizers in polymers opens prospects for their continuous and reusable application. Methylene blue (MB) and Rose Bengal were immobilized in polystyrene by mixing solutions of the photosensitizers in chloroform with a polymer solution, followed by air evaporation of the solvent. This procedure yielded 15–140 μm polymer films with a porous surface structure. The method chosen for immobilization ensured 99% enclosure of the photosensitizer in the polymer. The antimicrobial activity of the immobilized photosensitizers was tested against Gram‐positive and Gram‐negative bacteria. It was found that both immobilized photosensitizers exhibited high antimicrobial properties, and caused by a 1.5–3 log₁₀ reduction in the bacterial concentrations to their total eradication. The bactericidal effect of the immobilized photosensitizers depended on the cell concentration and on the illumination conditions. Scanning electron microscopy was used to prove that immobilized photosensitizers excited by white light caused irreversible damage to microbial cells. Photosensitizers immobilized on a solid phase can be applied for continuous disinfection of wastewater bacteria.     

Apoptosis induction and mitochondria alteration in human HeLa tumour cells by photoproducts of Rose Bengal acetate

The aim of this work was to investigate the apoptosis induction and mitochondria alteration after photodamage exerted by incubation of HeLa cells with Rose Bengal acetate-derivative (RBAc) followed by irradiation for a total dose of 1.6 J/cm2. This treatment was previously demonstrated to reduce cell viability under mild treatment conditions, suggesting the restoration of the photoactive molecule in particularly sensitive cell sites. Indeed, Rose Bengal (RB) is a very efficient photosensitizer, whose photophysical properties are inactivated by addition of the quencher group acetate. The RBAc behaves as a fluorogenic substrate by entering easily the cells where the original, photoactive molecule is restored by specific esterases. Different intracellular sites of photodamage of RB are present. In particular, fluorescence imaging of Rodamine 123 and JC-1 labelled cells showed altered morphology and loss of potential membrane of mitochondria. MTT and NR assays gave indications of alteration of mitochondrial and lysosomal enzyme activities. These damaged sites were likely responsible for triggering apoptosis. Significant amount of apoptotic cell death (about 40%) was induced after light irradiation followed RBAc incubation as revealed by morphological (modification of cell shape and blebs formation), cytochemical (FITC-Annexin-V positive cells) and nuclear fragmentation assays.     

Porphyrin-cellulose nanocrystals: a photobactericidal material that exhibits broad spectrum antimicrobial activity

Towards our overall objectives of developing potent antimicrobial materials to combat the escalating threat to human health posed by the transmission of surface-adhering pathogenic bacteria, we have investigated the photobactericidal activity of cellulose nanocrystals that have been modified with a porphyrin-derived photosensitizer (PS). The ability of these previously synthesized porphyrin-cellulose-nanocrystals (CNC-Por (1)) to mediate bacterial photodynamic inactivation was investigated as a function of bacterial strain, incubation time and illumination time. Despite forming an insoluble suspension, CNC-Por (1) showed excellent efficacy toward the photodynamic inactivation of Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii (MDRAB) and methicillin-resistant Staphylococcus aureus (MRSA), with the best results achieving 5-6 log units reduction in colony forming units (CFUs) upon illumination with visible light (400-700 nm; 118 J cm(-2)). CNC-Por (1) mediated the inactivation of Pseudomonas aeruginosa, although at reduced activity (2-3 log units reduction). Confocal laser scanning microscopy of CNC-Por (1) after incubation with A. baumannii or S. aureus suggested a lack of internalization of the PS. Research into alternative materials such as CNC-Por (1) may lead to their application in hospitals and healthcare-related industries wherein novel materials with the capability of reducing the rates of transmission of a wide range of bacteria, particularly antibiotic resistant strains, are desired.     

Systematic study of parameters influencing the action of Rose Bengal with visible light on bacterial cells: comparison between the biological effect and singlet-oxygen production

As part of a project to study different methods for the disinfection of effluent water, the inactivation of different microorganisms (Escherichia coli, Deinococcus radiodurans and spores of Bacillus subtilis) using a combination of a photosensitizer (Rose Bengal) with simulated sunlight and oxygen was determined under various environmental conditions (temperature, pH index). In parallel, the singlet-oxygen (1O2) production was also measured under the same conditions. Whereas the vegetative cells could be inactivated much more efficiently at increased temperature and altered index of pH, the production of 1O2 remained essentially the same under these alterations. Additionally, the relations among the sensitivities of different cell types to be killed by our photodynamic treatments (PDT) were opposite to those found after exposure to ionizing radiation. The results of photodynamic experiments do not reflect the cells' capacity to repair DNA strand breaks. Spores of B. subtilis, as a nonvegetative system, could not be inactivated by illuminations up to 100 J cm-2. Together, these findings indicate that DNA is not the primary target, the inactivation of which leads to the killing of our test organisms. Instead, the cellular envelope appears to be the component being assaulted by our PDT.     

Selective Examination of Plasma Membrane–Associated Photosensitizers Using Total Internal Reflection Fluorescence Spectroscopy: Correlation between Photobleaching and Photodynamic Efficacy of Protoporphyrin IX

Fluorescence spectra, fluorescence decay kinetics, photobleaching kinetics and photodynamic efficacy of protoporphyrin IX (PP) were investigated in endothelial cells in vitro after different incubation times. Fluorescence spectra and photobleaching kinetics were determined during total internal reflection (TIR) illumination or epiillumination. Because penetration depth of the excitation light during TIR illumination was limited to about 100 nm, plasma membrane-associated PP was almost selectively examined. Spectra obtained by TIR fluorescence spectroscopy (FS) showed a very low background, where-as spectra obtained by epi-illumination exhibited considerable background by autofluorescence and scattered light. For photobleaching kinetics during TIR illumination after 1 h or 24 h incubation, a biexponential fluorescence decrease was observed with a rapidly and a slowly bleaching portion. After 1 h incubation, the rapidly bleaching portion was the predominant fraction, whereas after 24 h incubation comparable relative amounts of the rapidly and slowly bleaching portion were determined. The rapidly and slowly bleaching portion were assigned to PP monomers and aggregated species in close vicinity to the plasma membrane. Fluorescence decay measurements after epi-illumination support the decrease of PP monomers within the whole cell with increasing incubation time. In contrast to TIR illumination, photobleaching of PP during epi-illumination was characterized by slow monoexponential fluorescence decrease after 1 h or 24 h incubation. Photodynamic efficacy of PP using epi-illumination was found to depend strongly on incubation time. Considerable cell inactivation was determined for short incubation times (1 h or 3 h), whereas photodynamic efficacy was diminished for longer incubation times. Reduced photodynamic efficacy after long incubation times was assigned to the lower amount of photodynamically active monomers determined close to the plasma membrane as well as within the whole cell. In conclusion, TIRFS measurements are suggested to be an appropriate tool for the examination of the plasma membrane-associated photosensitizer fraction in living cells.     

Analyzing and monitoring of phage–bacteria interaction using CE

The utilization of CE for monitoring bacteria–phage interaction was investigated in this study. Streptococcus thermophilus and Lactobacillus bulgaricus strains and their phages were used as model bacteria and phages for the purpose of validation in this study. CE with heterogeneous polymer polyethylene oxide was utilized for the separation of intact bacteria and investigation of phage–bacteria interaction. An intact phage detection was carried out with CZE by adding SDS in the running buffer. Calibration graphs of bacteria and phages were obtained with R² values of 0.963 and 0.937, respectively. S. thermophilus strain was infected with its virulent phage B3‐X18 for investigation of phage–bacteria interaction. It was observed in capillary electropherogram that the culture was lysed depending on the multiplicity of infection value and it showed to be completely lysed when the multiplicity of infection value was 10. The interaction of S. thermophilus strain with L. bulgaricus phage was also investigated by using a CE and a microbiological method and it was observed that the L. bulgaricus phage attached itself to the cell wall of S. thermophilus strain without damaging the cell.     

MEMBRANE PHOTOMODIFICATION OF CARDIAC MYOCYTES: POTASSIUM AND LEAKAGE CURRENTS

Cardiac myocytes were isolated from the atria of frogs (Rana pipiens) and whole cell potassium (IK) and "leakage" (Ileak) currents were monitored using the patch clamp technique. Cells were photosensitized by exposure to Rose Bengal (0.125-0.5 microM). Illumination produced an exponential decrease in IK, and an increase in Ileak. Current modifications varied with light intensity and sensitizer concentration. IK stabilized when illumination ceased, while Ileak continued to increase at a slower rate after illumination ended. The exponential nature of IK modification suggests that potassium channels are photomodified with single hit kinetics. The stabilization of IK following illumination suggests (1) that the photomodification of the potassium channel does not involve long lasting (minutes) radical chain reactions and (2) that this photomodification is not repaired in the course of a few minutes.     

Microbiological Decontamination of Water: Improving the Solar Disinfection Technique (SODIS) with the Use of Nontoxic Vital Dye Methylene Blue

Rational use of water is a major challenge for governments and global organizations, with easy and inexpensive interventions being sought by communities that are not supplied with drinking water. In this context, solar disinfection (SODIS) has shown great efficiency for water disinfection. To speed up the process and improve inactivation, we studied the effects of methylene blue (MB) as a photodynamic agent because of its ability to absorb visible light (red wavelength) and generate singlet oxygen as a reactive species, thereby inactivating bacteria and viruses present in water. In this study, samples of clean mineral water were artificially contaminated with Gram‐positive (Staphylococcus epidermidis or Deinococcus radiodurans) or with Gram‐negative strains (Escherichia coli or Salmonella typhimurium) and exposed to traditional SODIS or to MB‐SODIS. A lethal synergistic effect was observed when cultures were illuminated in the presence of MB. The obtained results indicate that bacterial inactivation can be achieved in a much shorter time when using MB associated with SODIS treatment. Therefore, this technique was able to provide safe water for consumption through the inactivation of microorganisms in general, including pathogens and some strains resistant to the traditional SODIS procedure, thus allowing its use in areas usually less exposed to sunlight.     

Immobilized Rose Bengal as Photocatalyst for Metal-Free Thiocyanation of Azaheterocycles under Continuous Flow Conditions

The immobilization of Rose Bengal onto Merrifield resin and its application in the light-driven metal-free thiocyanation of azaheterocycles in continuo were investigated. The supported photocatalyst was used in batch reactions under heterogeneous conditions, with different azaheterocycles, and proved to efficiently promote the reaction. Its recovery and recycle were also demonstrated for up to four times. The resin-supported Rose Bengal was then employed under continuous flow conditions, affording thiocyanate functionalized azaheterocycles with higher space-time-yields than the in-batch transformations, and in a shorter reaction time. The easily modification of the SCN groups was also demonstrated by synthetizing trifluoromethyl thioethers and sulfenyl tetrazoles derivates in high yields.     

细菌变异株生长热谱研究

用微量热法研究微生物的生长与代谢,是生物热力学一个新的研究领域.我们已经测得细菌生长的完整热谱,这种热谱具有良好的重现性和明显的特征性,可以作为细菌的“指纹图”对其进行种的分类和鉴定[1～5].但在研究中发现,同种细菌不同变异株的生长图谱略有差异,若培养基选择?..     

First unambiguous evidence for distinct ionic and surface-contact effects during photocatalytic bacterial inactivation on Cu–Ag films: Kinetics,mechanism and energetics

Highly uniform Cu–Ag polyurethane (PU) sputtered films inactivated Escherichia coli presenting normal cell wall porins but were shown to require longer times to inactivate genetically modified porinless Escherichia coli. The Cu–Ag–PU films were observed to inactivate E. coli ～7 times faster compared to Cu-films and ～15 times faster compared to Ag-films by themselves under low intensity actinic light irradiation. The galvanic contact between Ag and Cu induces atomic disorder in the film leading to corrosion and release of ppb amounts of Ag and Cu. This is suggested to induce the oligodynamic effect responsible for the bacterial inactivation on the Cu–Ag films. By ATR-FTIR spectroscopy, the structural changes in the E. coli (LPS) bilayer were monitored during bacterial inactivation for porin and porinless bacteria. The Ag₂Cu₂O₄ composite was found by X-ray photoelectron spectroscopy (XPS) to be the predominant species in the Cu–Ag films leading to bacterial inactivation. A mechanism is suggested for the photo-induced interfacial charge transfer (IFCT) between both metal oxides.