Photodynamic treatment with hexyl-aminolevulinate mediates reversible thiol oxidation in core oxidative stress signaling proteins.

Photodynamic therapy (PDT) is a highly selective two-step cancer treatment involving a photosensitizer and illumination with visible light in the presence of molecular oxygen. PDT is clinically approved worldwide for treating several premalignant conditions and cancer forms, especially endoscopically accessible tumors and dermatological malignancies. PDT-mediated cytotoxicity takes place via autophagy, apoptosis and necrosis, but the exact trigger mechanisms for various death-pathways are still unknown. PDT induces reactive oxygen species (ROS) through photochemical reactions. ROS can react with different macromolecules resulting in cellular damage, including oxidation of proteins. One of the known protein modifications is reversible oxidation of cysteine thiols (-SH), which in many cases constitute a redox switch to modulate protein activity and cellular signaling. Here we have examined the role of reversible oxidation of protein thiols as a potential mediator of cytotoxicity after hexylaminolevulinate-mediated photodynamic treatment (HAL-PDT) in the human epidermoid carcinoma cell line A431. Nearly 2300 proteins were found to be reversibly oxidized after HAL-PDT, of which 374 high-confidence proteins were further allocated to cellular compartments and functional networks. 115 of the high confidence proteins were associated with apoptosis and 257 have previously not been reported to be reversibly oxidized on cysteines. We find an enrichment of DNA damage checkpoint and oxidative stress response proteins. Many of these constitute potential signaling hubs in apoptosis, including ATM, p63, RSK1 p38, APE1/Ref-1 and three 14-3-3 family members. Our study represents the first comprehensive mapping of reversibly oxidized proteins subsequent to HAL-PDT. Several of the proteins constitute potentially novel redox-regulated apoptotic triggers as well as potential targets for adjuvants that may improve the efficacy of HAL-PDT and PDT using other photosensitizers.

A comparative study on the antibacterial photodynamic efficiency of a curcumin derivative and a formulation on a porcine skin model.

INTRODUCTION: The propagation of pathogens resistant to antibiotics around the globe has induced an urgent call for action: alternatives to conventional antibiotic therapy have to be developed to prevent a post-antibiotic catastrophe. This study focuses on the enhancement of Photodynamic Inactivation (PDI) of Gram(+) versus Gram(-) bacteria comparing a cationic derivative of curcumin (SACUR-3) to curcumin bound to polyvinylpyrrolidone (PVP-CUR) using an ex vivo porcine skin model to simulate an application on the human skin and foodstuff. EXPERIMENTAL: Porcine skin samples were inoculated with either Staphylococcus aureus or Escherichia coli and treated with either SACUR-3 or PVP-CUR at concentrations of 50 or 100 µM, respectively. Subsequent to blue light illumination (435 nm, 33.8 J cm(-2)) quantitative analyses were performed by counting the colony forming units. Furthermore, the localization of both photoactive compounds in the porcine skin was determined by fluorescence microscopy. PDI of S. aureus resulted in a reduction of 2.2 log10 steps if employing 50 µM of SACUR-3 and of 1.7 log10 steps with 50 µM of PVP-CUR. Phototoxicity towards E. coli was 3.3 log10 steps using 100 µM of SACUR-3 and 0.3 log10 steps for 100 µM of PVP-CUR. Both compounds do not exceed the stratum corneum of the skin. CONCLUSION: A direct comparison of both approaches yields that the cationic curcumin derivative SACUR-3 is effective against Gram(+) and Gram(-) pathogens, whereas the formulation of PVP-CUR has a photokilling effect on the Gram(+) model strain only, but leaves the approval of curcumin as a food additive E100 unaffected. Our results suggest the applicability of SACUR-3-based PDI in dermatology, hand hygiene and food production.

Methylsulfonyl Zn phthalocyanine: A polyvalent and powerful hydrophobic photosensitizer with a wide spectrum of photodynamic applications.

BACKGROUND: The biomedical photodynamic principle is based on the light-induced and photosensitizer-mediated killing of unwanted or harmful cells by overproduction of reactive oxygen species. Motivated by the success of photodynamic therapy (PDT) against several types of tumors, further applications of this approach are constantly identified which require the design and synthesis of novel photosensitizers with specifically tailored properties for a particular clinical application. METHODS: Hydrophobic photosensitizers are currently gaining attention and hence a tetramethylsulfonyl Zn(II) phthalocyanine (2) was designed with respect to the desired photoproperties. The photodynamic potential of 2 was assessed by the determination of its photophysical and photochemical properties, and by a large range of biological tests including its phototoxicity against cancer cells and Gram(+) bacteria. Unsubstituted ZnPc was used as a reference compound for comparison purposes. RESULTS: Phthalocyanine 2 has a better oxygen generation and is more photostable than ZnPc. 2 is a polyvalent and powerful hydrophobic photosensitizer with a wide spectrum of photodynamic applications against cancer (tested on A431 cells) and for Gram(+) PDI. Against Staphylococcus aureus, a maximum photokilling efficiency of more than 6 log10 steps was induced by a 5µM concentration of 2, outperforming the 3 log10 criterion for an antimicrobial effect (according to the recommendation of the American Society for Microbiology) by more than three orders of magnitude. CONCLUSIONS: Phthalocyanine 2 has attractive photophysical and -chemical characteristics. Initial evaluation of its application in anti-tumor PDT and PDI suggest potential for further pre-clinical and clinical development of this compound.

Photodynamic decontamination of foodstuff from Staphylococcus aureus based on novel formulations of curcumin.

Increasing antibiotic resistance is one of the world's greatest health problems. The food chain is an important factor in the transfer of resistant germs from animals to humans. This study focuses on photodynamic inactivation (PDI), employing curcumin bound to polyvinylpyrrolidone (PVP-C) and NovaSol®-curcumin as photosensitizers, as potent tool for the decontamination of cucumber, pepper and chicken meat from Staphylococcus aureus (serving as the model for methicillin-resistant S. aureus, MRSA). Both curcumin and PVP have been approved as food additives, consequently exhibiting excellent biocompatibility. Vegetables and meat were contaminated with S. aureus and sprinkled with PVP-C and NovaSol®-curcumin at concentrations of 50 and 100 µM, respectively. Illumination was performed immediately using visible light (435 nm, 9.4 mW cm(-2), 33.8 J cm(-2)). The PDI efficiency was determined by quantitative analyses of colony forming units 24 h post illumination. Additionally, the long-term effects of the photodynamic inactivation on cucumbers were investigated by quantitative analyses of the viable bacterial fraction after 24 and 48 h. Photodynamic inactivation of S. aureus revealed a mean reduction of 2.6 log10 (99.8%) for cucumbers, 2.5 log10 (99.7%) for pepper and 1.7 log10 (98%) for chicken meat relative to control samples. The bactericidal effect compared to controls seems to last for at least 48 h. Furthermore, no visible changes of the exterior appearance of foodstuff after photodynamic decontamination were observed. Photodynamic inactivation may therefore constitute a safe, economic and effective decontamination technique, which is harmless to health and not noticeable to consumers.

Back to the roots: photodynamic inactivation of bacteria based on water-soluble curcumin bound to polyvinylpyrrolidone as a photosensitizer.

Photodynamic inactivation (PDI), the light-induced and photosensitizer-mediated overproduction of reactive oxygen species in microorganisms, represents a convincing approach to treat infections with (multi-resistant) pathogens. Due to its favourable photoactive properties combined with excellent biocompatibility, curcumin derived from the roots of turmeric (Curcuma longa) has been identified as an advantageous photosensitizer for PDI. To overcome the poor water solubility and the rapid decay of the natural substance at physiological pH, we examined the applicability of polyvinylpyrrolidone curcumin (PVP-C) in an acidified aqueous solution (solubility of PVP-C up to 2.7 mM) for photoinactivation of Gram(+) and Gram(-) bacteria. Five micromolar PVP-C incubated for 5 minutes and illuminated using a blue light LED array (435 ± 10 nm, 33.8 J cm(-2)) resulted in a >6 log10 reduction of the number of viable Staphylococcus aureus. At this concentration, longer incubation periods result in a lower phototoxicity, most likely due to degeneration of curcumin. Upon an increase of the PVP-C concentration to 50 µM (incubation for 15 or 25 min) a complete eradication of Staphylococcus aureus can be achieved. As expected for a non-cationic photosensitizer, cell wall permeabilization with CaCl2 prior to addition of 50 µM PVP-C for 15 min is necessary to induce a drop in the count of the Gram(-) Escherichia coli for more than 3 log10. As both constituents of the formulation, curcumin (E number E100) and polyvinylpyrrolidone (E1201), have been approved as food additives, a PDI based on PCP-C might allow for a very sparing clinical application (e.g. for disinfection of wounds) or even for employment in aseptic production of foodstuffs.