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Cefadroxil is a widely used antibiotic with a low elimination efficiency in wastewater treatments plants, so it represents a contaminants of emerging concern that should be removed. The photosensitization process that involves natural pigments and visible sunlight can be offered as an environmentally friendly alternative to be considered for Cefadroxil degradation. In this investigation, we carried out a mechanistic and kinetic approach to Cefadroxil photodegradation sensitized by Riboflavin and Humic Acid, in individual and combined processes. Our experiments indicate that Cefadroxil is able to interact with the excited states of Riboflavin as well as with the photogenerated reactive oxygen species, with an important contribution of singlet oxygen. The antibiotic was less sensitive to the photodegradation in the presence of Humic Acids and in the mixture of Riboflavin and Humic Acids. Self-sensitization processes and internal filter effects are proposed as possible explanations for the observed phenomenon. The reaction between Cefadroxil and singlet oxygen showed a dependence with the pH of the medium, the photodegradation kinetic constants are greater at alkaline pH compared to neutral pH. The reaction is favored when the anionic species of the antibiotic is present. Microbiological tests on S. aureus indicated that the antibiotic reduce its antimicrobial activity as a consequence of the photooxidative process mediated by singlet oxygen. We believe that the results are relevant since, the sensitized photodegradation process could lead to the oxidation of Cefadroxil and to the progressive loss of its antimicrobial function, this fact could contribute to the decrease in the generation of bacterial multi-resistance to antibiotics in the environment., (© 2024. The Author(s), under exclusive licence to the European Photochemistry Association, European Society for Photobiology.)

The effect of photodynamic inactivation (PDI) sensitized by 5,10,15,20-tetra(4-N,N,N-trimethylammoniophenyl)porphyrin (TMAP 4+ ) on different components of mono- and dual-species biofilms of Staphylococcus aureus and Escherichia coli was determined by different methods. First, the plate count technique showed that TMAP 4+ -PDI was more effective on S. aureus than E. coli biofilm. However, crystal violet staining revealed no significant differences between before and after PDI biofilms of both bacteria. On the other hand, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method indicated a reduction in viable cells as the light exposure time increases in both, mono- and dual-species biofilms. Furthermore, it was determined that as the irradiation time increases, the amount of extracellular polymeric substances present in the biofilms decreased. This effect was presented in both strains and in the mixed biofilm, being more evident in S. aureus mono-specie biofilm. Finally, scanning electron microscopy analysis showed a decrease in the number of cells forming the biofilm after photosensitization treatments. This information makes it possible to determine whether the photodynamic action is based on damage to metabolic activity, extracellular matrix and/or biomass, which may be useful in establishing a fully effective PDI protocol for the treatment of microorganisms growing as biofilms., (© 2024. The Author(s), under exclusive licence to the European Photochemistry Association, European Society for Photobiology.)

To improve bacterial photodynamic inactivation (PDI), this work analyzes the photodynamic effect caused by the combination of photosensitizers (PSs) on two bacterial models and different growth mode. Simultaneous administration of PSs from different families, zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc 4+ ), 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin (TMAP 4+ ), meso-tetrakis(9-ethyl-9-methyl-3-carbazoyl)chlorin (TEMCC 4+ ) and 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl] chlorin (TAPC) was investigated against Staphylococcus aureus and Escherichia coli, in planktonic form, biofilm and growth curve. Various PSs combinations showed greater inactivation compared to when used separately under the same conditions but at twice the concentration. However, differences were found in the effectiveness of the PSs combinations on Gram positive and negative bacteria, as well as in planktonic or biofilm form. Likewise, the combination of three PSs completely stopped E. coli growth under optimal nutritional conditions. PSs combination allows extending the range of light absorption by agents that absorb in different areas of the visible spectrum. Therefore, PDI with combined PSs increases its antimicrobial capacity using agents' concentrations and light fluences lower than those necessary to cause the same effect as single PS. These advances represent a starting point for future research on the potentiation of PDI promoted by the combined use of PSs., (© 2023. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

Phthalocyanine derivatives have been proposed as photosensitizers for the treatment of several microbial infections. In this review, the progress in the structures of phthalocyanines was analyzed, considering that these compounds can easily functionalize and can form complexes with various metal ions. In this sense, different substituents were used to increase the interaction with the microorganisms, improving their photodynamic inactivation. Furthermore, these photosensitizers absorb strongly at phototherapeutic window, emit red fluorescence, and efficiently produce the formation of reactive oxygen species. Subsequently, the influence of binding, bacteria types, cell density, washing effect, and media on photoinactivation was remarked to elimination of microbes. Finally, photokilling of bacterial biofilm by phthalocyanines and the mechanism of action were discussed. Therefore, this review brings together the main features of phthalocyanines as antimicrobial phototherapeutic agents., (© 2022 The Chemical Society of Japan & Wiley-VCH GmbH.)

The main causes of food spoilage come from the process of oxidation and the contamination by microorganisms. For the purpose of increasing food shelf-life the industries employ different techniques, being the addition of preservatives, one of the most used. The aim of this contribution was to investigate the potential antioxidant properties of tyrosol (4-hydroxyphenethyl alcohol, 4-OH) and tyrosol derived isomers (2-hydroxyphenethyl alcohol, 2-OH and 3-hydroxyphenethyl alcohol, 3-OH) against reactive oxygen species (ROS) and the antimicrobial effect on Staphylococcus aureus and Escherichia coli. Furthermore, the type of antioxidant effect of substrates and commercial antioxidants mixtures was studied. Upon visible-light, the substrates interacted with the vitamin B2 and different ROS were generated. All the compounds deactivated O 2 ( 1 Δ g ) and O 2 ●- , whereas only 2-OH and 3-OH inhibited H 2 O 2 and HO ● . The substrates exhibited a synergistic antioxidant effect when combined with commercial antioxidants. 2-OH showed antimicrobial activity against strains tested., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Photodynamic inactivation (PDI) protocols using photoactive metallated porphyrin-doped conjugated polymer nanoparticles (CPNs) and blue light were developed to eliminate multidrug-resistant pathogens. CPNs-PDI protocols using varying particle concentrations and irradiation doses were tested against nine pathogenic bacterial strains including antibiotic-resistant bacteria of the ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species ) pathogens group. The bactericidal effect was achieved in methicillin-resistant Staphylococus aureus ( S. aureus ) strains using low light doses (9.6-14.4 J/cm 2 ), while Gram-negative bacteria required a higher light dose (28.8 J/cm 2 ). The bacteria-CPN interaction was studied through flow cytometry, taking advantage of the intrinsic CPN fluorescence, demonstrating that CPNs efficiently bind to the bacterial envelope. Finally, the performance of CPNs-PDI was explored in biofilms; good antibiofilm ability and almost complete eradication were observed for S. aureus and Escherichia coli biofilms, respectively, using confocal microscopy. Overall, we demonstrated that CPNs-PDI is an efficient tool not only to kill superbugs as sessile cells but also to disrupt and eradicate biofilms of highly relevant pathogenic bacterial species.

The development of convenient synthetic methods and improved materials for the production of high load-capacity and biocompatible drug delivery systems is a challenging task with important implications in health sciences. In this work, acrylamide/2-hydroxyethylmethacrylate and N-isopropylacrylamide/2-hydroxyethylmethacrylate hydrogels were synthesized by photopolymerization using energy-efficient green-LEDs. A functionalized silsesquioxane was used as both crosslinker and co-initiator for the photopolymerization. The hybrid organic-inorganic nature of the silsesquioxane improved the resulting hydrogels' properties increasing their swelling capacity and biocompatibility. Additionally, the mild conditions used during the photopolymerization allowed the synthesis of hydrogels in the presence of antibiotics yielding high load-capacity materials in which the drug preserves its molecular structure and antimicrobial activity (as confirmed by HPLC and microbiological assays). The materials were characterized by FTIR, DSC and SEM. Additionally, the kinetics of gels´ swelling and drug release were studied under physiological conditions (pH 7.4 and 37 °C). The results demonstrate how hydrogel composition affects the antibiotics-release kinetics. The final drug release percentage increased with increasing molar fraction of acrylamide or N-isopropylacrylamide and in most cases exceeded 85%. Finally, the antibacterial effect of loaded gels was characterized using a number of assays against Gram negative and Gram positive bacteria. The observed antibacterial effect correlated well with swelling and drug release results. Furthermore, gels are not toxic for isolated erythrocytes as demonstrated by haemolytic tests. Overall, our results indicate that the produced hydrogels are promising materials to develop controlled drug-delivery devices such as capsules, dermatological patches and others., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The goal of this work was to investigate the photodynamic activity of 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]chlorin (TAPC) and zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine iodide (ZnPPc 4+ ) as photosensitizers to inactivate Staphylococcus aureus biofilms and prevent their formations in different culture media., Methods: We incubated S aureus biofilms in different culture media: tryptic soy (TS), nutrient (N), Müeller Hinton (MH) broth, TS with glucose 2 and 5% (w/v) with 5 μM ZnPPc 4+ or TAPC and irradiated with visible light (350-800 nm). Photodynamic inactivation (PDI) was determined by count of colony forming units (CFU) and crystal violet method. Furthermore, we studied PDI effect on biofilm development in TS broth. Finally, we examined the effects of PDI on the structure of S aureus biofilm., Results: Greater inactivation was achieved, using TAPC or ZnPPc 4+ , when S aureus biofilm was grown in N or MH broths rather than in TS. Besides, glucose addition to the medium decreases the ability to develop biofilm and increase the photoinactivation capacity. Prevention of 3 log biofilm developments was obtained when S aureus cultures were treated with TAPC (10 μM) and 108 J/cm 2 in TS broth and the number of CFU was counted after 24 hours. Moreover, microscopy studies demonstrated modifications in biofilm architecture., Conclusions: These results indicate that TAPC and ZnPPc 4+ may be promising photosensitizers for photodynamic inactivation of S aureus biofilms or to prevent their formation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The photodynamic action mechanism sensitized by a non-charged porphyrin-fullerene C 60 dyad (TCP-C 60 ) and its tetracationic analogue (TCP-C 60 4+ ) was investigated in solution and in Staphylococcus aureus cells. The ability of both dyads to form a photoinduced charge-separated state was evidenced by the reduction of methyl viologen in N , N -dimethylformamide (DMF). Moreover, the formation of superoxide anion radicals induced by these dyads was detected by the reduction of nitro blue tetrazolium. Also, photosensitized decomposition of l-tryptophan (Trp) was investigated in the presence of reactive oxygen species (ROS) scavengers. The addition of β-carotene and sodium azide had a slight effect on reaction rate. However, photooxidation of Trp mediated by TCP-C 60 was negligible in the presence of d-mannitol, while no protection was found using TCP-C 60 4+ . In a polar medium, these dyads mainly act by a contribution of type I pathway with low generation of singlet molecular oxygen, O 2 ( 1 Δ g ). In S. aureus cell suspensions, an aerobic atmosphere was required for the photokilling of this bacterium. The photocytotoxicity induced by TCP-C 60 was increased in D 2 O with respect to water, while a small effect was found using TCP-C 60 4+ . Furthermore, photoinactivation of microbial cells was negligible in the presence of sodium azide. The addition of d-mannitol did not affect the photoinactivation induced by TCP-C 60 . In contrast, S. aureus cells were protected by d-mannitol when TCP-C 60 4+ was used as a photosensitizer. Also, generation of O 2 ( 1 Δ g ) in the S. aureus cells was higher for TCP-C 60 than TCP-C 60 4+ . Therefore, TCP-C 60 appears to act in microbial cells mainly through the mediation of O 2 ( 1 Δ g ). Although, a contribution of the type I mechanism was found for cell death induced by TCP-C 60 4+ . Therefore, these dyads with high capacity to produce photoinduced charge-separated state represent interesting photosensitizers to inactivate microorganisms by type I or type II mechanisms. In particular, TCP-C 60 may be located in a non-polar microenvironment in the cells favoring a type II pathway, while a contribution of the type I mechanism was produced using the cationic TCP-C 60 4+ ., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Sensitized photooxidation processes in the presence of natural pigments may provide an alternative to antibiotics degradation since these compounds are transparent to natural light irradiation, therefore, they can be degraded by the action of photosensitizers which absorb light and produce highly reactive species, especially those derived from molecular oxygen (ROS). Most antibiotics used currently belong to a group of pharmaceutical substances that have been considered a new type of contaminants due to their persistence and bioaccumulation in the environment., Objective: In this context, we decided to investigate the kinetic and mechanistic aspects of Vancomycin (Vanco) photosensitized degradation in the presence of the natural pigment Riboflavin (Vitamin B2, Rf) and the artificial dye Rose Bengal (RB) for comparative purposes., Methods: The study have been done by using Stationary photolysis, Laser flash photolysis, Time-resolved phosphorence detection of O 2 (1Δg) experiments and Bactericidal activity evaluation. The experiments were carried out in aqueous solution at different pH values in order to establish relationships between the structure of the compound and its susceptibility to ROS-mediated photooxidation., Results: Experimental evidence indicates that in the presence of Rf there is considerable contribution of the radical-mediated mechanism, while in the presence of RB the photooxidation process occurs exclusively through O 2 (1Δg) and the reactivity to this excited species increases with increasing pH of the environment., Discussion: The results obtained, have been shown that Rf can raise the photodegradation of Vanco by both the radical pathway and the O 2 ( 1 Δ g ) mediated. Furthermore, the antibiotic is able to interact with the excited electronic states of Rf as well as O 2 ( 1 Δ g ) generated by energy transfer between the excited triplet state of the photosensitizer and the oxygen ground state. The predominant mechanism for photodegradation of Vanco in the presence of the Rf is the radical via because of the considerable interaction with the excited triplet state of the photosensitizer demonstrated by laser flash photolysis experiments. Microbiological test on Staphylococcus aureus ATCC25923 showed that the bactericidal activity of the antibiotic on the strain studied was affected by the sensitized photodegradation process, suggesting that photoproducts generated eventually do not retain the bactericidal properties of the original antibiotic.

The photodynamic mechanism sensitized by N,N-dimethyl-2-[4-(3-N,N,N-trimethylammoniopropoxy)phenyl]fulleropyrrolidinium (DPC 60 2+ ) was investigated in Staphylococcus aureus cells. Different experimental conditions were used to detect reactive oxygen species (ROS) in S. aureus cell suspensions. First, a photoinactivation of 4 log decrease of S. aureus viability was chosen using 0.5μM DPC 60 2+ and 15min irradiation. An anoxic atmosphere indicated that oxygen was required for an effective photoinactivation. Also, photoprotection was found in the presence of sodium azide, whereas the photocytotoxicity induced by DPC 60 2+ increased in D 2 O. The addition of diazabicyclo[2.2.2]octane or d-mannitol produced a reduction in the S. aureus photokilling. Moreover, singlet molecular oxygen, O 2 ( 1 Δ g ), was detected by the reaction with 9,10-dimethylanthracene into the S. aureus cells. A decrease in the photoinactivation of S. aureus was observed in the presence of β-nicotinamide adenine dinucleotide reduced form, which was dependent on the NADH concentration. Therefore, under aerobic condition the photocytotoxicity activity induced by DPC 60 2+ was mediated by mainly a contribution of type II process. Moreover, photoinactivation of S. aureus was possible with DPC 60 2+ in the presence of azide anions under anoxic condition. However, these conditions were not effective to photoinactivate Escherichia coli. On the other hand, the addition of potassium iodide produced an increase in the photokilling of bacteria, depending on the KI concentration and irradiation times. The formation of reactive iodine species may be contributing to inactivate S. aureus cells photoinduced by DPC 60 2+ ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A novel 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]chlorin (TAPC) was synthesized by reduction of the corresponding porphyrin TAPP with p-toluenesulfonhydrazide, followed by selective oxidation with o-chloranil. Spectroscopic properties and the photodynamic activity of these photosensitizers were compared in N,N-dimethylformamide. An increase in the absorption band at 650nm was found for the chlorin derivative with respect to TAPP. These photosensitizers emit red fluorescence with quantum yields of 0.15. Both compounds were able to photosensitize singlet molecular oxygen with quantum yields of about 0.5. Also, the formation of superoxide anion radical was detected in the presence of TAPC or TAPP and NADH. Photodynamic inactivation was investigated on a Gram-positive bacterium Staphylococcus aureus, a Gram-negative bacterium Escherichia coli and a fungal yeast Candida albicans cells. In vitro experiments showed that TAPC or TAPP were rapidly bound to microbial cells at short incubation periods. These photosensitizers, without intrinsic positive charges, contain four basic amino groups. These substituents can be protonated at physiological pH, increasing the interaction with the cell envelopment. Photosensitized inactivation improved with an increase of both photosensitizer concentrations and irradiation times. After 15min irradiation, a 7 log reduction of S. aureus was found for treated with 1μM photosensitizer. Similar result was obtained with E. coli after using 5μM photosensitizer and 30min irradiation. Also, the last conditions produced a decrease of 5 log in C. albicans cells. Therefore, TAPC was highly effective as a broad-spectrum antimicrobial photosensitizer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A porphyrin-fullerene C60 dyad (TCP-C60) substituted by carbazoyl groups was used to obtain electrogenerated polymeric films on optically transparent indium tin oxide (ITO) electrodes. This approach produced stable and reproducible polymers, holding fullerene units. The properties of this film were compared with those formed by layers of TCP/TCP-C60 and TCP/ZnTCP. Absorption spectra of the films presented the Soret and Q bands of the corresponding porphyrins. The TCP-C60 film produced a high photodecomposition of 2,2-(anthracene-9,10-diyl)bis(methylmalonate), which was used to detect singlet molecular oxygen O2((1)Δg) production in water. In addition, the TCP-C60 film induced the reduction of nitro blue tetrazolium to diformazan in the presence of NADH, indicating the formation of superoxide anion radical. Moreover, photooxidation of L-tryptophan mediated by TCP-C60 films was found in water. In biological media, photoinactivation of Staphylococcus aureus was evaluated depositing a drop with 2.5 × 10(3) cells on the films. After 30 min irradiation, no colony formation was detected using TCP-C60 or TCP/TCP-C60 films. Furthermore, photocytotoxic activity was observed in cell suspensions of S. aureus and Escherichia coli. The irradiated TCP-C60 film produced a 4 log decrease of S. aureus survival after 30 min. Also, a 4 log reduction of E. coli viability was obtained using the TCP-C60 film after 60 min irradiation. Therefore, the TCP-C60 film is an interesting and versatile photodynamic active surface to eradicate bacteria.

The effect of divalent cations, EDTA and chitosan (CS) on the uptake and photoinactivation of Escherichia coli produced by 5,10,15,20-tetrakis(4-N,N,N-trimethylammoniumphenyl)porphyrin (TMAP(4+)), 5,10-di(4-methylphenyl)-15,20-di(4-N,N,N-trimethylammoniumphenyl)porphyrin (MPAP(2+)) and 5,10,15,20-tetra(4-sulphonatophenyl)porphyrin (TPPS(4-)) were examined under different conditions. These porphyrins were rapidly bound to E. coli cells (<2.5min) and the uptake of photosensitizers was not dependent on incubation temperature, reaching values of 0.61, 0.18 and 0.08nmol/10(8) cells for TMAP(4+), MPAP(2+) and TPPS(4-), respectively. The addition of Ca(2+) or Mg(2+) to the cultures enhanced the uptake of MPAP(2+) and TPPS(4-) by cells. In contrast, the amount of TMAP(4+) bound to cells was decreased. The presence of EDTA produced an increase in the uptake of porphyrins by cells, while CS mainly enhanced the amount of TPPS(4-) bound to E. coli. The photoinactivation of E. coli cells mediated by TMAP(4+) was highly effective even at low concentration (1μM) and short irradiation period (5min). However, a reduction in the phototoxicity was found for TMAP(4+) in presence of Ca(2+) and Mg(2+). In contrast, the phototoxic activity mediated by MPAP(2+) and TPPS(4-) was increased. Addition of EDTA did not show effect on the photoinactivation induced by cationic porphyrins, while a small enhance was found for TPPS(4-). Moreover, inactivation of E. coli cells was achieved in the presence CS. This cationic polymer was antimicrobial by itself in the dark. Using a slightly toxic CS concentration, the phototoxic activity induced by TMAP(4+) was diminished. This effect was mainly observed at lower concentration of TMAP(4+) (0.5-1μM). In contrast, an increase in E. coli photoinactivation was obtained for MPAP(2+) and TPPS(4-) in presence of CS. Thus, this natural polymeric destabilizer agent mainly benefited the photoinactivation mediated by TPPS(4-)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Trace amounts of the widely used β-lactam antibiotics (Atbs) in waste water may cause adverse effects on the ecosystems and contribute to the proliferation of antibiotic-resistant bacteria. On these grounds, kinetic and mechanistic aspects of photosensitized degradation of Ceftriaxone (Cft) and Cefotaxime (Ctx), have been studied in pure water by stationary and time-resolved techniques. Additionally, possible implications of these photoprocesses on the antimicrobial activity of the Atbs have also been investigated. Photoirradiation of aqueous solutions of Cft and Ctx produces the degradation of both Atbs in the presence of Riboflavin (vitamin B2), a well known pigment dissolved in natural aquatic systems. The process occurs through Type I and Type II mechanisms, with effective prevalence of the former. The participation of O2(-), OH and O2((1)Δg) is supported by experiments of oxygen consumption carried out in the presence of specific scavengers for such reactive oxygen species. Microbiological assays exhibit a parallelism between the rate of Cft and Ctx photodegradation and the loss of their bactericidal capacity on Staphylococcus aureus strains. Results contribute to both understanding kinetic and mechanism aspects of the degradation and predicting on natural decay of Atbs waste water-contaminants., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A covalently linked porphyrin-fullerene C60 dyad 5 was synthesized by 1,3-dipolar cycloaddition using 5-(4-formylphenyl)-10,15,20-tris[3-(N-ethylcarbazoyl)]porphyrin, N-methylglycine and fullerene C60. Methylation of 5 was used to obtain a cationic dyad 6. Spectroscopic properties were compared in toluene, N,N-dimethylformamide (DMF) and toluene/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water reverse micelles. Absorption spectra of the dyads were essentially a superposition of the spectra of the porphyrin and fullerene reference compounds, indicating a very weak interaction between the chromophores in the ground state. The fluorescence emission of the porphyrin moiety in the dyads was strongly quenched by the attached fullerene C60 unit. The singlet molecular oxygen, O2((1)Δg), productions (ΦΔ) were strongly dependent on the solvent polarity. Similar ΦΔ values were obtained for 5,10,15,20-tetrakis[3-(N-ethylcarbazoyl)]porphyrin (TCP) in both solvents. Also, dyad 5 showed a high O2((1)Δg) generation in toluene. However, O2((1)Δg) production mediated by 5 considerably diminished in the more polar solvent DMF. Also, a high photodynamic activity involving O2((1)Δg) was found for both dyads in a simple biomimetic system formed by AOT reverse micelles. The photoinactivation ability of these dyads was investigated in Staphylococcus aureus cell suspensions. Photosensitized inactivation of S. aureus by dyad 6 exhibits a 4.5 log decrease of cell survival (99.997% cell inactivation), when the cultures are treated with 5 μM photosensitizer and irradiated with visible light (350-800 nm) for 30 min. Under these conditions, a lower photocytotoxic effect was found for 5 (3.2 log decrease). Furthermore, photoinactivation induced by 6 was higher than those obtained with the separate moieties of the dyad. Therefore, molecular structures formed by porphyrin-fullerene C60 dyads represent interesting photosensitizers to inactivate S. aureus., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

The photodynamic mechanism of action induced by N,N-dimethyl-2-(4'-N,N,N-trimethylaminophenyl)fulleropyrrolidinium iodide (DTC60(2+)) was investigated on Candida albicans and Escherichia coli cells. First, photogeneration of superoxide anion radical by DTC60(2+) in the presence of NADH was detected using nitro blue tetrazolium method in reverse micelles. In C. albicans suspensions, 10 μM DTC60(2+) was an effective photosensitizer, producing a ∼5log decrease of cell survival when the cultures were irradiated for 30 min with visible light. Also, C. albicans cells growth was not detected in the presence of 10 μM DTC60(2+) and irradiation. Photodynamic mechanism investigations were compared in both C. albicans and E. coli cells. Studies under anoxic conditions indicated that oxygen was required for the photodynamic inactivation of these microorganisms. The photocytotoxicity induced by DTC60(2+) was similar in D2O than in water cell suspensions. Furthermore, photoinactivation of microbial cells was negligible in the presence of azide ion, while the addition of mannitol produced a photoprotective effect on the cellular survival. These results indicate that DTC60(2+) has potential as agent to the photodynamic inactivation of microbial cells. Also, the photocytotoxicity activity induced by this cationic fullerene derivative can involve the intermediacy of both superoxide anion radical and singlet molecular oxygen., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Photoinactivation of Streptococcus mitis induced by zinc(II) 2,9,16,23-tetrakis[2-(N,N,N-trimethylamino)ethoxy]phthalocyanine (ZnEPc(4+)) was studied under different experimental condition in order to obtain information about the photodynamic processes and the cellular damage. A 3 log decrease in S. mitis survival was found in cell suspensions (~2×10(8) cells/mL) incubated with 2 μM ZnEPc(4+) and irradiated for 30 min with visible light (54 J/cm(2)). Also, S. mitis cells growth was not detected in broth treated with 5 μM ZnEPc(4+) under continuous irradiation. Studies of photodynamic action mechanism showed that the cells were protected in the presence of azide ion, while the addition of mannitol did not produce a significant effect on the survival. Moreover, the photocytotoxicity was increased in D2O indicating the interference of singlet molecular oxygen. On the other hand, it was found that ZnEPc(4+) interacts strongly with calf thymus DNA in solution but photocleavage of DNA was only detected after long irradiation periods. After S. mitis photoinactivation, modifications of genomic DNA were not observed by electrophoresis. In contrast, the transmission electron microscopy showed structural changes in the S. mitis cells, exhibiting mesosome-like structures. After 2h irradiation, the cytoplasm showed segregation patterns and PDI appeared to have effects on the cell wall, including variability in wall thickness. Also, the presence of bubbles was detected on the cell surface by scanning electron microscopy. However, the photodamage to the cell envelope was insufficient to cause the release of intracellular biopolymers. Therefore, modifications in the cytoplasmic biomolecules and alteration in the cell barriers could be mainly involved in S. mitis photoinactivation. It can be concluded that photosensitization by ZnEPc(4+) mainly involved a type II photoprocess, while alteration in the cytoplasmatic components and modifications in the cell envelope were the major cause for the photoinactivation of S. mitis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodule formation on roots of alfalfa plants. S. meliloti produces two exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote symbiosis. EPS I and EPS II are secreted in two major fractions that reflect differing degrees of subunit polymerization, designated high- and low-molecular-weight fractions. We reported previously that EPSs are crucial for autoaggregation and biofilm formation in S. meliloti reference strains and isogenic mutants. However, the previous observations were obtained by use of "domesticated" laboratory strains, with mutations resulting from successive passages under unnatural conditions, as has been documented for reference strain Rm1021. In the present study, we analyzed the autoaggregation and biofilm formation abilities of native S. meliloti strains isolated from root nodules of alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all the native isolates revealed a high degree of identity with reference S. meliloti strains. PCR analysis of the expR gene of all the isolates showed that, as in the case of reference strain Rm8530, this gene is not interrupted by an insertion sequence (IS) element. A positive correlation was found between autoaggregation and biofilm formation abilities in these rhizobia, indicating that both processes depend on the same physical adhesive forces. Extracellular complementation experiments using mutants of the native strains showed that autoaggregation was dependent on EPS II production. Our results indicate that a functional EPS II synthetic pathway and its proper regulation are essential for cell-cell interactions and surface attachment of S. meliloti.

Novel unsymmetrically substituted Zn(II) phthalocyanine bearing an adamantylethoxy group (AZnPc) was synthesized by the ring expansion reaction of boron(III) subphthalocyanine chloride with an appropriated phthalonitrile derivative (APc). Also, APc was used to obtain a new Zn(II) phthalocyanine bearing four adamantylethoxy groups (A(4)ZnPc) by cyclotetramerization reaction. The spectroscopic and photodynamic properties of these photosensitizers were compared with those of a Zn(II) phthalocyanine substituted by four methoxy groups (M(4)ZnPc) in different media. Similar results were obtained in N,N-dimethylformamide. However, a higher photodynamic activity was found for AZnPc in a biomimetic system formed by reverse micelles. This behavior was also observed in the presence of human red blood (HRB) cells, which were used as an in vitro cellular model. Thus, AZnPc was the most effective photosensitizer to produce HRB cells hemolysis. The photodynamic effect produced a decrease in the HRB cells osmotic stability leading to the release of hemoglobin. Studies of photodynamic action mechanism showed that photohemolysis of HRB cells was protected in the presence of azide ion, while the addition of mannitol produced a negligible effect on the cellular photodamage, indicating the intermediacy of O(2)((1)Δ(g)). Therefore, the presence of an adamantyl unit in the phthalocyanine macrocycle represents an interesting molecular architecture for potential phototherapeutic agents., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

The photodynamic inactivation (PDI) of Escherichia coli and Streptococcus mitis sensitized by cationic phthalocyanines was studied in different media containing blood derivatives. First, the activity of zinc(II) tetramethyltetrapyridino[3,4-b:3',4'-g:3'',4''-l:3''',4'''-q]porphyrazinium (ZnAPc4+), zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc4+) and zinc(II) 2,9,16,23-tetrakis[2-(N,N,N-trimethylamino)ethoxy]phthalocyanine (ZnEPc4+) were compared to photoinactivate these bacteria in saline solutions. After visible light irradiation, a higher photoinactivation of E. coli cells was found for ZnPPc4+, while ZnEPc4+ was the more effective sensitizer to eradicate S. mitis cells. In the presence of human red blood (HRB) cells, two aspects were analyzed: the photohemolysis induced by these cationic phthalocyanines and the PDI of bacteria in medium containing erythrocytes. The highest photohemolytic damage was produced by ZnPPc4+, which can be avoided using azida ion as photoprotective quencher. In both bacteria, the photoinactivation is possible in presence of HRB cells. Mainly, ZnEPc4+ is effective to photoinactivate S. mitis with a low hemolysis of erythrocytes. However, inactivation of E. coli by ZnPPc4+ decreases in medium with HRB cells, further when azide ion is added to avoid hemolysis. The presence of plasma considerable reduces the photocytotoxic effect, which mainly affects the eradication of E. coli. However, the PDI of S. mitis by ZnEPc4+ is even possible in presence of blood derivatives., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

Photodynamic inactivation (PDI) of Escherichia coli has been studied in cultures treated with zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc(+4)) to obtain insight about the mechanism of damage. This phthalocyanine is rapidly bound to cells, reaching a value of approximately 0.8 nmol/10(6) cells when the cultures were incubated with 2 microM sensitizer. After 30 min of irradiation, a 4 log decrease of E. coli survival was observed. The photocytotoxic action was investigated in plasmid and genomic DNA by electrophoretic analysis. Absorption spectroscopic studies showed that this cationic phthalocyanine interacts strongly with DNA (K(DNA)=4.7 x 10(6)M(-1)). Photocleavage of calf thymus DNA sensitized by ZnPPc(+)4 was not found even after long irradiation periods. Similar results were also observed in genomic DNA extracted from E. coli cells after PDI treatment. Modifications of plasmid DNA isolated from bacteria were only observed after long irradiation periods. However, under these conditions transmission electron microscopy of the PDI bacteria revealed an aggregation of cytoplasmic macromolecules and irregularities in cell barriers. Also, scanning electron microscopy showed a shrunken appearance in cells after PDI. Even so, release of intracellular biopolymers was not detected by absorption. On the other hand, outer and inner membranes permeabilization assays showed an increase in the permeability. Consequently, alterations in the cell membrane functionality induced by ZnPPc(+4) appear to be the major cause of E. coli inactivation upon PDI.

The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been investigated in bacteria treated with 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) and visible light. The photosensitization activity of A3B3+ porphyrin was compared with that of 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin p-tosylate (TMAP4+), which is an active tetracationic sensitizer to eradicate bacteria. The PDI damages on plasmid and genomic DNA were analyzed by electrophoresis. DNA photocleavage was observed after a long period of irradiation, when the bacterial cells are largely photoinactivated. Transmission electron microscopy (TEM) revealed structural changes with appearance of low density areas into the cells and irregularities in cell barriers, which could affect the normal cell membrane functionality. Also, damages on the cell-wall were not detected by scanning electron microscopy (SEM) and release of intracellular biopolymers was not found after PDI. These results indicate that the photodynamic activity of these cationic porphyrins produces DNA photodamage after a long period of irradiation. Therefore, an interference with membrane functions could be the main cause of E. coli photoinactivation upon short PDI treatments.

A novel N,N-dimethyl-2-(4'-N,N,N-trimethylaminophenyl)fulleropyrrolidinium iodide (DTC(60)(2+)) has been synthesized by 1,3-dipolar cycloaddition using 4-(N,N-dimethylamino) benzaldehyde, N-methylglycine and fullerene C(60). This approach produced an N-methyl-2-(4'-N,N-dimethylaminophenyl)fulleropyrrolidine with 38% yield. Exhaustive methylation of this fullerene derivative with methyl iodide yielded 95% of dicationic DTC(60)(2+). The spectroscopic and photodynamic properties of the DTC(60)(2+) were compared with a non-charged N-methyl-2-(4'-acetamidophenyl)fulleropyrrolidine (MAC(60)) and a monocationic N,N-dimethyl-2-(4'-acetamidophenyl)fulleropyrrolidinium iodide (DAC(60)(+)). The dicationic DTC(60)(2+) is essentially aggregated in solution of different solvents and it is partially dissolved as monomer in benzene/benzyl-n-hexadecyldimethyl ammonium chloride (BHDC) 0.1M/water (W(0)=10) reverse micelles. The singlet molecular oxygen, O(2) ((1)Delta(g)), production was evaluated using 1,3-diphenylisobenzofuran. The photodynamic effect was strongly dependent on the medium, diminishes when the sensitizer is aggregated and increases in an appropriately surrounded microenvironment. The photodynamic inactivation produced by these fullerene derivatives was investigated in vitro on a typical Gram-negative bacterium, Escherichia coli. Photosensitized inactivation of E. coli cellular suspensions by DTC(60)(2+) exhibits a approximately 3.5 log decrease of cell survival (99.97% of cellular inactivation), when the cultures are treated with 1 microM of sensitizer and irradiated for 30 min. This photosensitized inactivation remains high even after one washing step. Also, the photodynamic activity was confirmed by growth delay of E. coli cultures. The growth was arrested when E. coli was exposed to 2 microM of cationic fullerene and irradiated, whereas a negligible effect was found for the non-charged MAC(60). These studies indicate that dicationic DTC(60)(2+) is an interesting agent with potential applications in photodynamic inactivation of bacteria.

The photodynamic effect of novel cationic porphyrins, with different pattern of meso-substitution by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl (A) and 4-(trifluoromethyl)phenyl (B) groups, have been studied in both solution bearing photooxidizable substrates and in vitro on a typical Gram-negative bacterium Escherichia coli. In these sensitizers, the cationic groups are separated from the macrocycle ring by a propoxy spacer. Thus, the charges have a high mobility and a minimal influence on photophysical properties of the porphyrin. These compounds produce singlet molecular oxygen, O2(1Delta(g)), with quantum yields of approximately 0.41-0.53 in N,N-dimethylformamide. In methanol, the l-tryptophan photodecomposition increases with the number of cationic charges in the sensitizer. In vitro investigations show that cationic porphyrins are rapidly bound to E. coli cells in approximately 5 min. A higher binding was found for A3B3+ porphyrin, which is tightly bound to cells still after three washing steps. Photosensitized inactivation of E. coli cellular suspensions follows the order: A3B3+ > A44+>> ABAB2+ > AB3+. Under these conditions, a negligible effect was found for 5,10,15,20-tetra(4-sulfonatophenyl)porphyrin (TPPS4(4-)) that characterizes an anionic sensitizer. Also, the results obtained for these new cationic porphyrins were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin (TTAP4+), which is a standard active sensitizer established to eradicate E. coli. The photodynamic activity of TTAP4+ is quite similar to that produced by A4(4+). Studies in an anoxic condition indicate that oxygen is necessary for the mechanism of action of photodynamic inactivation of bacteria. The higher photodynamic activity of A3B3+ was confirmed by growth delay experiments. Photodynamic inactivation capacities of these sensitizers were also evaluated in E. coli cells immobilized on agar surfaces. Under these conditions, A3B3+ porphyrin retains a high activity to inactivate localized bacterial cells. Therefore, tricationic porphyrin A3B3+ is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria in liquid suspensions or on surfaces.

The photodynamic effect of meso-substituted cationic porphyrins, 5-[4-(trimethylammonium)phenyl]-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin iodide 1, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 2 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were compared in N,N-dimethylformamide. Fluorescence quantum yields (varphiF) of 0.10, 0.06 and 0.08 were calculated for porphyrins 1, 2 and 3, respectively. The singlet molecular oxygen, O2(1Deltag), production was evaluated using 9,10-dimethylanthracene yielding values of 0.66, 0.36 and 0.42 for porphyrins 1, 2 and 3, respectively. Guanosine 5'-monophosphate was used as biological substrate model. Similar decomposition of guanosine 5'-monophosphate was obtained using these cationic porphyrins as sensitizer. In biological medium, photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. E. coli cultures were treated with sensitizer at 37 degrees C for 30 min in dark. In both procedures, a higher photoinactivation of cells (>99.999%) was found for cells treated with 10 microM of tricationic porphyrin 3 and irradiated for 5 min with visible light. Porphyrins 1 and 2 only show an important photodamage when the cells are irradiated without washing step. These results indicated that the tetracationic porphyrin 3 could be a promising sensitizer with potential applications in the photoinactivation of bacterial cells by photodynamic therapy.