Wolfgang Bäumler

The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria

New antibacterial strategies are required in view of the increasing resistance of bacteria to antibiotics. One promising technique involves the photodynamic inactivation of bacteria. Upon exposure to light, a photosensitizer in bacteria can generate singlet oxygen, which oxidizes proteins or lipids, leading to bacteria death. To elucidate the oxidative processes that occur during killing of bacteria, Staphylococcus aureus was incubated with a standard photosensitizer, and the generation and decay of singlet oxygen was detected directly by its luminescence at 1,270 nm. At low bacterial concentrations, the time-resolved luminescence of singlet oxygen showed a decay time of 6 ± 2 μs, which is an intermediate time for singlet oxygen decay in phospholipids of membranes (14 ± 2 μs) and in the surrounding water (3.5 ± 0.5 μs). Obviously, at low bacterial concentrations, singlet oxygen had sufficient access to water outside of S. aureus by diffusion. Thus, singlet oxygen seems to be generated in the outer cell wall areas or in adjacent cytoplasmic membranes of S. aureus. In addition, the detection of singlet oxygen luminescence can be used as a sensor of intracellular oxygen concentration. When singlet oxygen luminescence was measured at higher bacterial concentrations, the decay time increased significantly, up to ≈40 μs, because of oxygen depletion at these concentrations. This observation is an important indicator that oxygen supply is a crucial factor in the efficacy of photodynamic inactivation of bacteria, and will be of particular significance should this approach be used against multiresistant bacteria.

Absorption and fluorescence spectroscopic investigation of indocyanine green

Abstract Absorption spectra, fluorescence quantum distributions, fluorescence quantum yields and degrees of fluorescence polarization vs. dye concentration were determined for indocyanine green in methanol, water and aqueous albumin solution. The monomer fluorescence quantum yield is limited to a few per cent by internal conversion. In water, dimerization starts at low concentrations (approximately 3 × 10 −8 m ol dm −3 ) and lowers the fluorescence quantum yield. The strong affinity of the dye to albumin shifts the onset of dimerization to higher concentrations. In methanol, dimerization is weak and closely spaced pair formation dominates at high concentrations. Dye adsorption to albumin and dye aggregation in the solvents were analysed experimentally and theoretically.

Fast and effective skin ablation with an Er:YAG laser: Determination of ablation rates and thermal damage zones

Er:YAG lasers are known to superficially ablate skin and other tissues with minimal thermal coagulation zones. The ablation efficacy and thus the clinical applicability of these lasers, however, was limited due to small beam diameters and repetition rates. Aim of this study was to determine the ablation efficacy and the amount of thermal damage with a new high‐power high‐repetition‐rate Er:YAG laser and to find optimal treatment parameters for skin ablation.

INDOCYANINE GREEN: INTRACELLULAR UPTAKE AND PHOTOTHERAPEUTIC EFFECTS IN VITRO

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.

Light-Induced Decomposition of Indocyanine Green

PURPOSE To investigate the light-induced decomposition of indocyanine green (ICG) and to test the cytotoxicity of light-induced ICG decomposition products. METHODS ICG in solution was irradiated with laser light, solar light, or surgical endolight. The light-induced decomposition of ICG was analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. Porcine retinal pigment epithelial (RPE) cells were incubated with the light-induced decomposition products of ICG, and cell viability was measured by trypan blue exclusion assay. RESULTS Independent of the light source used, singlet oxygen (photodynamic type 2 reaction) is generated by ICG leading to dioxetanes by [2+2]-cycloaddition of singlet oxygen. These dioxetanes thermally decompose into several carbonyl compounds. The decomposition products were identified by mass spectrometry. The decomposition of ICG was inhibited by adding sodium azide, a quencher of singlet oxygen. Incubation with ICG decomposition products significantly reduced the viability of RPE cells in contrast to control cells. CONCLUSIONS ICG is decomposed by light within a self-sensitized photo oxidation. The decomposition products reduce the viability of RPE cells in vitro. The toxic effects of decomposed ICG should be further investigated under in vivo conditions.

PHOTOSTABILITY AND THERMAL STABILITY OF INDOCYANINE GREEN

The photo-fading of the S0-S1 absorption band of the infrared dye indocyanine green sodium iodide (ICG-NaI) has been studied by cw laser excitation to the S1 band. Monomeric solutions in water, heavy water, aqueous sodium azide, human plasma, methanol and dimethyl sulfoxide (DMSO) as well as J-aggregated solutions in H2O and D2O have been investigated. A leucoform of indocyanine green seems to be formed by photodegradation. The degradation slows down with exposure time. The initial degradation yield, phi D,0, is determined. In monomeric and dimeric water, heavy water and sodium azide solutions the initial photostability is of the order of phi D.0 approximately 10(-3), in the organic solvents methanol and DMSO it is of the order of phi D.0 approximately 10(-5), and in human plasma it is phi D.0 approximately 2 x 10(-6). J-aggregates at high concentration are very stable. The thermal stability of the ICG-NaI solutions at room temperature in the dark is compared with their photostability. The thermal degradation time of monomeric and dimeric ICG-NaI in water, heavy water and sodium azide solutions is t(th) approximately 10 days, while no thermal degradation is observed for ICG-NaI J-aggregates and ICG-NaI in methanol, DMSO and human plasma.

Endoscopic fluorescence detection of dysplasia in patients with Barrett's esophagus, ulcerative colitis, or adenomatous polyps after 5-aminolevulinic acid–induced protoporphyrin IX sensitization

BACKGROUND Surveillance of patients with Barretts esophagus or ulcerative colitis for dysplasia is confined to biopsy specimens taken randomly during endoscopy because dysplasia remains undetectable by visual inspection. We attempted to visualize dysplastic tissue during endoscopy after sensitization with 5-aminolevulinic acid (5-ALA) leading to accumulation and formation of protoporphyrin IX and induction of characteristic red fluorescence of the latter substance using blue light illumination. METHODS Six patients with histologically proven low- or high-grade dysplasia (Barretts esophagus 2, ulcerative colitis 1, Billroth-II stomach 1, rectal polyps 2) were treated with oral administration of different concentrations of 5-ALA (10 to 20 mg/kg) or by local instillation of 3 gm 5-ALA in the rectum. Endoscopic fluorescence detection was performed 1 to 6 hours after sensitization using a blue light source and compared with conventional white light endoscopy. Biopsies of fluorescent and nonfluorescent areas were compared with histologic findings. RESULTS Normal duodenal mucosa and squamous epithelium showed more intense 5-ALA-induced background red fluorescence compared with normal mucosa in the stomach or Barretts mucosa. Histologically, dysplasia was exclusively found in areas with red fluorescence. False-positive fluorescence was associated with microscopic inflammation of the mucosa or feces in the colon. CONCLUSIONS 5-ALA-induced protoporphyrin IX fluorescence may be useful in the detection of dysplasia in the gastrointestinal tract by enhancement of endoscopic surveillance of patients at a high risk for dysplasia.

Indocyanine green (ICG) and laser irradiation induce photooxidation

Abstract The cellular uptake and subcellular localization of indocyanine green (ICG; absorption band 700– 850 nm), and cell survival and ultrastructural changes following ICG-mediated phototherapy were investigated in vitro in four different cell lines derived from human skin (SCL1 and SCL2 squamous cell carcinoma, HaCaT keratinocytes and N1 fibroblasts). The cellular uptake of ICG (1–50 μ M , incubation times 1, 4, 24 h) was saturable, highly cumulative and could be inhibited by the addition of 250 μ M bromosulphophthalein indicating the involvement of the organic anion transporting polypeptide (OATP). For HaCaT cells, the maximum cellular uptake (V max ) and the Michaelis constant (K m ) were 9.9 ± 1.1 m M and 47 ± 16 μ M , respectively, following a 24-h incubation with ICG. Fluorescence microscopy revealed a cytoplasmic distribution of ICG, probably bound to glutathione S -transferase. Following irradiation with a cw-diode laser (805 nm, 80 mW/cm 2 ) at doses of 24 or 48 J/cm 2 , the phototoxicity was determined using the MTT assay as a measure of cell viability. For all cell lines, ICG concentrations above 25 μ M produced a significant phototoxic effect. The EC 50 of ICG for HaCaT cells following irradiation at 24 J/cm 2 was 20.1 ± 3.9 μ M . Growth curves showed that even HaCaT cells treated at the EC 50 were killed within a week following treatment. Electron microscopy 1 h after ICG-mediated phototherapy revealed cytoplasmic vesiculation, dilation of the rough endoplasmic reticulum, the Golgi complex and the perinuclear cisternae and the beginning of chromatin condensation in the nucleus. These ultrastructural findings are not consistent with a photothermal action of ICG-mediated phototherapy. Taken together with those of previous studies by our group these results support photooxidation as a major cell-killing mechanism.

Q-switch laser and tattoo pigments: first results of the chemical and photophysical analysis of 41 compounds.

In the Western world, there are at least 20–30 million people with tattoos. Improved self‐image and social stigmatization are the main reasons for removing tattoos from skin. Q‐switched lasers are applied to destroy the tattoo compounds in the skin. The treatment of tattoos containing ink often gives excellent results, whereas the results of treatments for coloured tattoos are not predictable and usually are worse. The chemical structure and the absorption spectra of the tattoo pigments are usually unknown. However, the efficacy of the treatment by using light of different Q‐switched lasers (wavelengths 510, 532, 694, 755, 1064 nm) is correlated to both the chemical structure of the tattooed compounds yielding specific absorption spectra and the laser wavelength used.

Long-pulse dye laser for photodynamic therapy: investigations in vitro and in vivo.

Continuous wave lasers or incoherent lamps are used effectively for photodynamic therapy (PDT). As the mechanism of action of pulsed lasers in PDT is not known, we investigated the efficacy of PDT with 5‐aminolevulinic acid (ALA) using a long‐pulse (1.5 ms) tunable flashlamp‐pumped pulsed dye laser (LPDL) in vitro and in vivo.