Xavier Ragàs

Photodynamic inactivation of Acinetobacter baumannii using phenothiazinium dyes: in vitro and in vivo studies.

Phenothiazinium dyes have been reported to be effective photosensitizers inactivating a wide range of microorganisms in vitro after illumination with red light. However, their application in vivo has not extensively been explored. This study evaluates the bactericidal activity of phenothiazinium dyes against multidrug‐resistant Acinetobacter baumannii both in vitro and in vivo.

Cationic Porphycenes as Potential Photosensitizers for Antimicrobial Photodynamic Therapy

Structures of typical photosensitizers used in antimicrobial photodynamic therapy are based on porphyrins, phthalocyanines, and phenothiazinium salts, with cationic charges at physiological pH values. However, derivatives of the porphycene macrocycle (a structural isomer of porphyrin) have barely been investigated as antimicrobial agents. Therefore, we report the synthesis of the first tricationic water-soluble porphycene and its basic photochemical properties. We successfully tested it for in vitro photoinactivation of different Gram-positive and Gram-negative bacteria, as well as a fungal species (Candida) in a drug-dose and light-dose dependent manner. We also used the cationic porphycene in vivo to treat an infection model comprising mouse third degree burns infected with a bioluminescent methicillin-resistant Staphylococcus aureus strain. There was a 2.6-log(10) reduction (p < 0.001) of the bacterial bioluminescence for the PDT-treated group after irradiation with 180 J·cm(-2) of red light.

Singlet oxygen in Escherichia coli: New insights for antimicrobial photodynamic therapy

Antimicrobial photodynamic therapy is an emerging treatment for bacterial infections that is becoming increasingly more attractive because of its effectiveness and unlikelihood of inducing bacterial resistance. However, there is limited knowledge about the localization of the photoactive drug in the bacteria and about the details of production of the main cytotoxic species, singlet oxygen. This article describes a combination of spectroscopic and time-resolved photophysical techniques that provide such information for a cationic porphyrin photosensitizer in gram-negative Escherichia coli bacteria. Our results reveal a double localization of the photosensitizer, inside (bound to the nucleic acids) and outside (bound to the cell wall) of the E. coli cells. Singlet oxygen is produced at both sites and is able to cross the cell wall.

Synthesis, characterization, and photoinduced antibacterial activity of porphyrin-type photosensitizers conjugated to the antimicrobial peptide apidaecin 1b.

Antimicrobial photodynamic therapy (aPDT) is an emerging treatment for bacterial infections that is becoming increasingly more attractive because of its effectiveness against multi-antibiotic-resistant strains and unlikelihood of inducing bacterial resistance. Among the strategies to enhance the efficacy of PDT against Gram-negative bacteria, the binding to a cationic antimicrobial peptide offers the attractive prospect for improving both the water solubilty and the localization of the photoactive drug in bacteria. In this work we have compared a number of free and apidaecin-conjugated photosensitizers (PSs) differing in structure and charge. Our results indicate that the conjugation of per se ineffective highly hydrophobic PSs to a cationic peptide produces a photosensitizing agent effective against Gram-negative bacteria. Apidaecin cannot improve the phototoxic activity of cationic PSs, which mainly depends on a very high yield of singlet oxygen production in the surroundings of the bacterial outer membrane. Apidaecin-PS conjugates appear most promising for treatment protocols requiring repeated washing after sensitizer delivery.

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

Fluorescent proteins are increasingly becoming actuators in a range of cell biology techniques. One of those techniques is chromophore-assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is useful to avoid uncontrolled photodamage, although the inactivation mechanisms by ROS are dependent on the fluorescent protein and are not fully understood. Herein, we present a quantitative study of the ability of the red fluorescent protein TagRFP to produce ROS, in particular singlet oxygen ((1)O(2)). TagRFP is able to photosensitize (1)O(2) with an estimated quantum yield of 0.004. This is the first estimation of a quantum yield of (1)O(2) production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime compared to EGFP, which reflects relatively high oxygen accessibility to the chromophore. The insight into the structural and photophysical properties of TagRFP has implications in improving fluorescent proteins for fluorescence microscopy and CALI.

Singlet oxygen in antimicrobial photodynamic therapy: Photosensitizer-dependent production and decay in E. coli

Several families of photosensitizers are currently being scrutinized for antimicrobial photodynamic therapy applications. Differences in physical and photochemical properties can lead to different localization patterns as well as differences in singlet oxygen production and decay when the photosensitizers are taken up by bacterial cells. We have examined the production and fate of singlet oxygen in Escherichia coli upon photosensitization with three structurally-different cationic photosensitizers, namely New Methylene Blue N (NMB), a member of the phenothiazine family, ACS268, a hydrophobic porphyrin with a single cationic alkyl chain, and zinc(II)-tetramethyltetrapyridinoporphyrazinium salt, a phthalocyanine-like photosensitizer with four positive charges on the macrocycle core. The kinetics of singlet oxygen production and decay indicate different localization for the three photosensitizers, whereby NMB appears to localize in an aqueous-like microenvironment, whereas ACS268 localizes in an oxygen-shielded site, highly reactive towards singlet oxygen. The tetracationic zinc(II) tetrapyridinoporphyrazine is extensively aggregated in the bacteria and fails to produce any detectable singlet oxygen.

Singlet oxygen photosensitisation by GFP mutants: oxygen accessibility to the chromophore

Aiming at the rational development of genetically-encoded photosensitisers, the production of singlet oxygen has been assessed for a number of class-2 Green Fluorescent Protein (GFP) mutants by means of time-resolved near-infrared luminescence detection. The accessibility of molecular oxygen to the chromophore seems to play a major role in the ability of GFPs to photosensitise singlet oxygen and this can be modulated by introducing specific mutations such as replacement of His148 by a less bulky amino acid. GFPs are also good singlet oxygen quenchers, hence further developments in this area should also seek to eliminate those amino acids with the highest quenching ability, particularly those at the protein surface and in the vicinity of the chromophore.

Monitoring of singlet oxygen luminescence and mitochondrial autofluorescence after illumination of hypericin/mitochondria complex: a time-resolved study

A study of hypericin (Hyp) interaction with mitochondria isolated from U-87 MG glioma cells as well as the time-resolved measurement of singlet oxygen (1O2) formation and annihilation after illumination of the Hyp/mitochondria complex is presented in this work. Interaction between Hyp and mitochondria was studied by steady-state and time-resolved UV–vis absorption and fluorescence spectroscopy. A high concentration of Hyp leads to the aggregation of this compound inside the mitochondria and the relative population of the monomeric (biologically active) form of Hyp decreases concomitantly to approximately 10% at the highest used Hyp bulk concentration. Photosensitized production of 1O2 in mitochondria after illumination of the Hyp/mitochondria complex is characterized by a rise lifetime of ~8 μs and shows saturation behaviour with respect to Hyp concentration. The lifetime of 1O2 depends on the composition of the medium where the mitochondria are suspended, ranging from about 3.0 μs in pure water to 26 μs in H2O–D2O (1:9) phosphate buffer. Our results confirm that only the monomeric form of Hyp is able to produce its excited triplet state, which consequently leads to 1O2 production. An influence of photoactivated Hyp on the mitochondria respiration chain was evaluated by the monitoring of time-resolved NAD(P)H fluorescence. We have demonstrated the rise of the NAD(P)H content after illumination of the Hyp/mitochondria complex.

Solvent and media effects on the photophysics of naphthoxazole derivatives

The photophysical properties of 2‐phenyl‐naphtho[1,2‐d][1,3]oxazole, 2(4‐N,N‐dimethylaminophenyl)naphtho[1,2‐d][1,3]oxazole and 2(4‐N,N‐diphenylaminophenyl) naphtho[1,2‐d][1,3]oxazole were studied in a series of solvents. UV–Vis absorption spectra are insensitive to solvent polarity whereas the fluorescence spectra in the same solvent set show an important solvatochromic effect leading to large Stokes shifts. Linear solvation energy relationships were employed to correlate the position of fluorescence spectra maxima with microscopic empirical solvent parameters. This study indicates that important intramolecular charge transfer takes place during the excitation process. In addition, an analysis of the solvatochromic behavior of the UV–Vis absorption and fluorescence spectra in terms of the Lippert–Mataga equation shows a large increase in the excited‐state dipole moment, which is also compatible with the formation of an intramolecular charge‐transfer excited state. We propose both naphthoxazole derivatives as suitable fluorescent probes to determine physicochemical microproperties in several systems and as dyes in dye lasers; consequence of their high fluorescence quantum yields in most solvents, their large molar absorption coefficients, with fluorescence lifetimes in the range 1–3 ns as well as their high photostability.