Johannes Regensburger

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 21st 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.

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.

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

Please cite this paper as: Tattoo inks contain polycyclic aromatic hydrocarbons that additionally generate deleterious singlet oxygen. Experimental Dermatology 2010; 19: e275–e281.

2D luminescence imaging of physiological wound oxygenation

Abstract:  In cutaneous wound healing, the role of oxygen in vivo is poorly understood. We studied wound surface pO2 during physiological wound healing in humans. Split‐thickness skin graft donor sites (n = 12) served as standardized wound models. Wound surface pO2 was measured at 1, 6 and 14 days after split‐skin harvesting using two‐dimensional luminescence lifetime imaging (2D‐LLI) of palladium(II)‐meso‐tetraphenyl‐tetrabenzoporphyrin (Pd‐TPTBP) in polystyrene‐co‐acrylonitrile (PSAN) particles on transparent foils. In another experiment, we removed the stratum corneum (SC) on the volar forearm (n = 10) by tape strippings to study the impact of the SC on the epidermal oxygen barrier. Split‐skin donor site pO2 significantly decreased during the time course of physiological healing. Regional differences in pO2 within donor site wounds were visualized for the first time in literature. No difference was found in pO2 before and after SC removal, showing that the SC is not a major constituent of the epidermal oxygen barrier.

Improving photodynamic inactivation of bacteria in dentistry: highly effective and fast killing of oral key pathogens with novel tooth-colored type-II photosensitizers.

Increasing antibiotic resistances in microorganisms create serious problems in public health. This demands alternative approaches for killing pathogens to supplement standard treatment methods. Photodynamic inactivation of bacteria (PIB) uses light activated photosensitizers (PS) to generate reactive oxygen species immediately upon illumination, inducing lethal phototoxicity. Positively charged phenalen-1-one derivatives are a new generation of PS for light-mediated killing of pathogens with outstanding singlet oxygen quantum yield ΦΔ of >97%. Upon irradiation with a standard photopolymerizer light (bluephase C8, 1260 ± 50 mW/cm(2)) the PS showed high activity against the oral key pathogens Enterococcus faecalis, Actinomyces naeslundii, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans. At a concentration of 10 μM, a maximum efficacy of more than 6 log10 steps (≥ 99.9999%) of bacteria killing is reached in less than 1 min (light dose 50 J/cm(2)) after one single treatment. The pyridinium substituent as positively charged moiety is especially advantageous for antimicrobial action.

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.

Time dependence of singlet oxygen luminescence provides an indication of oxygen concentration during oxygen consumption

Singlet oxygen plays a major role in photodynamic inactivation of tumor cells or bacteria. Its efficacy depends critically on the oxygen concentration [O(2)], which can decrease in case oxygen is consumed caused by oxidative reactions. When detecting singlet oxygen directly by its luminescence at 1270 nm, the course of the luminescence signal is critically affected by [O(2)]. Thus, it should be feasible to monitor oxygen consumption during photo-oxidative processes. Singlet oxygen was generated by exciting a photosensitizer (TMPyP) in aqueous solution (H(2)O or D(2)O) of albumin. Chromatography shows that most of the TMPyP molecules are unbound, and therefore singlet oxygen molecules can diffuse in the solution. A sensor device for oxygen concentration revealed a rapid decrease of [O(2)] (oxygen depletion) in the solution during irradiation. The extent of oxygen depletion in aqueous albumin solution depends on the radiant exposure and the solvent. When detecting the luminescence signal of singlet oxygen, the shape of the luminescence signal significantly changed with irradiation time. Thus, local oxygen consumption could be monitored during photodynamic action by evaluating the course of singlet oxygen luminescence.

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.

The impact of absorbed photons on antimicrobial photodynamic efficacy.

Due to increasing resistance of pathogens toward standard antimicrobial procedures, alternative approaches that are capable of inactivating pathogens are necessary in support of regular modalities. In this instance, the photodynamic inactivation of bacteria (PIB) may be a promising alternative. For clinical application of PIB it is essential to ensure appropriate comparison of given photosensitizer (PS)-light source systems, which is complicated by distinct absorption and emission characteristics of given PS and their corresponding light sources, respectively. Consequently, in the present study two strategies for adjustment of irradiation parameters were evaluated: (i) matching energy doses applied by respective light sources (common practice) and (ii) by development and application of a formula for adjusting the numbers of photons absorbed by PS upon irradiation by their corresponding light sources. Since according to the photodynamic principle one PS molecule is excited by the absorption of one photon, this formula allows comparison of photodynamic efficacy of distinct PS per excited molecule. In light of this, the antimicrobial photodynamic efficacy of recently developed PS SAPYR was compared to that of clinical standard PS Methylene Blue (MB) regarding inactivation of monospecies biofilms formed by Enterococcus faecalis and Actinomyces naeslundii whereby evaluating both adjustment strategies. PIB with SAPYR exhibited CFU-reductions of 5.1 log10 and 6.5 log10 against E. faecalis and A. naeslundii, respectively, which is declared as a disinfectant efficacy. In contrast, the effect of PIB with MB was smaller when the applied energy dose was adjusted compared to SAPYR (CFU-reductions of 3.4 log10 and 4.2 log10 against E. faecalis and A. naeslundii), or there was even no effect at all when the number of absorbed photons was adjusted compared to SAPYR. Since adjusting the numbers of absorbed photons is the more precise and adequate method from a photophysical point of view, this strategy should be considered in further studies when antimicrobial efficacy rates of distinct PS-light source systems are compared.

Fatty acids and vitamins generate singlet oxygen under UVB irradiation

Abstract:  UVB radiation is already known as initiator and promoter of carcinogenesis in skin. UVB is well absorbed in proteins and DNA leading to products such as cyclobutane pyrimidine dimers. In contrast, UVA radiation generates reactive oxygen species such as singlet oxygen, which can initiate a variety of cellular damages and cellular signalling. It was the goal to investigate whether and to which extent UVB radiation is additionally able to cause oxidative damages via singlet oxygen. Potential endogenous photosensitizers such as vitamin B molecules or unsaturated fatty acids were irradiated in solution using monochromatic UVB radiation at 308 nm. Singlet oxygen was directly detected and quantified by its luminescence at 1270 nm. All investigated endogenous photosensitizers showed clear singlet oxygen signals with a quantum yield ranging from 5 to 40%. UVB radiation altered the photosensitizer molecules during irradiation yielding a change of absorption in the entire ultraviolet spectrum (280–400 nm). UVB irradiation of endogenous photosensitizers produced singlet oxygen that in turn changes the absorption of those molecules. Being an important prerequisite, the changed absorption may either reduce or increase singlet oxygen production. An increase in singlet oxygen generation may initiate a vicious cycle that has the potential to amplify UVB‐ or UVA‐mediated effects in skin cells.