Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells.

PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

Electron beam functionalized photodynamic polyethersulfone membranes - photophysical characterization and antimicrobial activity.

Polymer membranes are powerful filtration tools in medicine and water treatment. Their efficiency and operational lifetime is limited by biofouling caused by microorganisms. This study describes the development of photodynamical active antimicrobial polymer membranes in a one-pot functionalization step using a well-known photosensitizer (PS). Commercially available polyethersulfone (PES) membranes for microfiltration were doped with the polycationic PS TMPyP using electron beam irradiation. These membranes were characterized in terms of binding stability and quantification of the PS and membrane morphology. Furthermore, the photodynamic ability was verified by time resolved singlet oxygen luminescence scans and successfully tested against the Gram-negative bacterium E. coli under low dose white light illumination resulting in the reduction in cell survival of 6 log10 units. Finally, in preliminarily experiments the photodynamic action against the Gram-positive bacteria M. luteus and the Gram-negative P. fluorescence and the mold C. cladosporioides was demonstrated. These promising results show the high photodynamic potential of electron beam functionalization of PES membranes with TMPyP. It preserves the photodynamic abilities of the immobilized PS resulting in efficient photodynamic inactivation of bacteria and mold on the membrane surface. The uprising worldwide spread of antibiotic resistant bacteria makes the development of new antibacterial strategies an inevitable challenge. The photodynamic inactivation of bacteria and its adaptation for antimicrobial surfaces, e.g. filtration membranes for water treatment, displays many advantages in terms of a wide application range, low mutagenic potential and environmental compatibility.

Singlet oxygen luminescence kinetics under PDI relevant conditions of pathogenic dermatophytes and molds.

A treatment of onychomycosis using the photodynamic effect would be a favorable alternative to currently used antimycotic drugs. This study should be considered as a first step towards development and control of an efficient photodynamic inactivation of onychomycosis causative pathogens. Here, we evaluate the usage of time-resolved 2D singlet oxygen luminescence detection in combination with 2D fluorescence scanning as a tool to understand the behavior of the photosensitizer when applied to fungi on Petri dishes. To investigate the interaction of photosensitizer with fungi in various concentrations and in different stages of live, a photodynamic inactivation was avoided by keeping the samples in darkness. Scans of singlet oxygen luminescence and photosensitizer fluorescence were performed over a period of 24days. Two different photosensitizer, a cationic porphyrin and cationic corrole and two fungi strains, the dermatophyte Trichophyton rubrum and the mold Scopulariopsis brevicaulis, were investigated in this study. The two-dimensional correlation of photosensitizer fluorescence and singlet oxygen luminescence revealed differences in the diffusion of both photosensitizer. Even though the singlet oxygen luminescence was quenched with increasing growth of fungi, it was found that the kinetics of singlet oxygen luminescence could be detected on Petri dishes for both photosensitizers and both fungi strains for up to seven days.

Synthesis, Photophysics and PDT Evaluation of Mono-, Di-, Tri- and Hexa-PEG Chlorins for Pointsource Photodynamic Therapy.

Pointsource photodynamic therapy (PSPDT) is a newly developed fiber optic method aimed at the delivery of photosensitizer, light and oxygen to a diseased site. Because of a need for developing photosensitizers with desirable properties for PSPDT, we have carried out a synthetic, photophysical and phototoxicity study on a series of PEGylated sensitizers. Chlorin and pheophorbide sensitizers were readily amenable to our synthetic PEGylation strategy to reach triPEG and hexaPEG galloyl pheophorbides and mono-, di-, triPEG chlorins. On screening these PEG sensitizers, we found that increasing the number of PEG groups, except for hexaPEGylation, increases phototoxicity. We found that three PEG groups but not less or more were optimal. Of the series tested, a triPEG gallyol pheophorbide and a triPEG chlorin were the most efficient at generating singlet oxygen, and produced the highest phototoxicity and lowest dark toxicity to Jurkat cells. A detailed kinetic analysis of the PEGylated sensitizers in solution and cell culture and media is also presented. The data provide us with steps in the development of PSPDT to add to the PDT tools we have in general.

In vivo photoacoustic tumor tomography using a quinoline-annulated porphyrin as NIR molecular contrast agent.

The synthesis and photophysical properties of a tetra-PEG-modified and freely water-soluble quinoline-annulated porphyrin are described. We previously demonstrated the ability of quinoline-annulated porphyrins to act as an in vitro NIR photoacoustic imaging (PAI) contrast agent. The solubility of the quinoline-annulated porphyrin derivative in serum now allowed the assessment of the efficacy of the PEGylated derivative as an in vivo NIR contrast agent for the PAI of an implanted tumor in a mouse model. A multi-fold contrast enhancement when compared to the benchmark dye ICG could be shown, a finding that could be traced to its photophysical properties (short triplet lifetimes, low fluorescence and singlet oxygen sensitization quantum yields). A NIR excitation wavelength of 790 nm could be used, fully taking advantage of the optical window of tissue. Rapid renal clearance of the dye was observed. Its straight-forward synthesis, optical properties with the possibility for further optical fine-tuning, nontoxicity, favorable elimination rates, and contrast enhancement make this a promising PAI contrast agent. The ability to conjugate the PAI chromophore with a fluorescent tag using a facile and general conjugation strategy was also demonstrated.

Development of Singlet Oxygen Luminescence Kinetics during the Photodynamic Inactivation of Green Algae.

Recent studies show the feasibility of photodynamic inactivation of green algae as a vital step towards an effective photodynamic suppression of biofilms by using functionalized surfaces. The investigation of the intrinsic mechanisms of photodynamic inactivation in green algae represents the next step in order to determine optimization parameters. The observation of singlet oxygen luminescence kinetics proved to be a very effective approach towards understanding mechanisms on a cellular level. In this study, the first two-dimensional measurement of singlet oxygen kinetics in phototrophic microorganisms on surfaces during photodynamic inactivation is presented. We established a system of reproducible algae samples on surfaces, incubated with two different cationic, antimicrobial potent photosensitizers. Fluorescence microscopy images indicate that one photosensitizer localizes inside the green algae while the other accumulates along the outer algae cell wall. A newly developed setup allows for the measurement of singlet oxygen luminescence on the green algae sample surfaces over several days. The kinetics of the singlet oxygen luminescence of both photosensitizers show different developments and a distinct change over time, corresponding with the differences in their localization as well as their photosensitization potential. While the complexity of the signal reveals a challenge for the future, this study incontrovertibly marks a crucial, inevitable step in the investigation of photodynamic inactivation of biofilms: it shows the feasibility of using the singlet oxygen luminescence kinetics to investigate photodynamic effects on surfaces and thus opens a field for numerous investigations.

Photodynamic inactivation of biofilm building microorganisms by photoactive facade paints.

This study was performed as a proof of concept for singlet oxygen generating facade paint as an alternative to conventional biocide containing facade paint for the prevention of biofilm growth on outdoor walls. Biofilms on outdoor walls cause esthetic problems and economic damage. Therefore facade paints often contain biocides. However commercially available biocides may have a series of adverse effects on living organisms as well as harmful environmental effects. Furthermore, biocides are increasingly designed to be more effective and are environmentally persistent. Thus, an eco-friendly and non-harmful to human health alternative to conventional biocides in wall color is strongly recommended. The well-known photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) was used as an additive in a commercially available facade paint. The generation of singlet molecular oxygen was shown using time resolved 2D measurements of the singlet oxygen luminescence. The photodynamic activity of the photosensitizer in the facade paint was demonstrated by phototoxicity tests with defined mold fungi and a mixture of microorganisms harvested from native outdoor biofilms as model organisms. It was proven in general that it is possible to inhibit the growth of biofilm forming microorganisms growing on solid wall paint surfaces by the cationic photosensitizer TMPyP added to the facade paint using daylight conditions for illumination in 12h light and dark cycles.