Porphyrin-BODIPY Dyad: Enhancing Photodynamic Inactivation via Antenna Effect.

A porphyrin-BODIPY dyad (P-BDP) was obtained through covalent bonding, featuring a two-segment design comprising a light-harvesting antenna system connected to an energy acceptor unit. The absorption spectrum of P-BDP resulted from an overlap of the individual spectra of its constituent parts, with the fluorescence emission of the BODIPY unit experiencing significant quenching (96 %) due to the presence of the porphyrin unit. Spectroscopic, computational, and redox investigations revealed a competition between photoinduced energy and electron transfer processes. The dyad demonstrated the capability to sensitize both singlet molecular oxygen and superoxide radical anions. Additionally, P-BDP effectively induced the photooxidation of L-tryptophan. In suspensions of Staphylococcus aureus cells, the dyad led to a reduction of over 3.5 log (99.99 %) in cell survival following 30 min of irradiation with green light. Photodynamic inactivation caused by P-BDP was also extended to the individual bacterium level, focusing on bacterial cells adhered to a surface. This dyad successfully achieved the total elimination of the bacteria upon 20 min of irradiation. Therefore, P-BDP presents an interesting photosensitizing structure that takes advantage of the light-harvesting antenna properties of the BODIPY unit combined with porphyrin, offering potential to enhance photoinactivation of bacteria.

Role of curvature in acridone for 1 O2 oxidation of a natural product homoallylic alcohol: A novel iso-hydroperoxide intermediate.

A density functional theoretical (DFT) study is presented, implicating a 1 O2 oxidation process to reach a dihydrobenzofuran from the reaction of the natural homoallylic alcohol, glycocitrine. Our results predict an interconversion between glycocitrine and an iso-hydroperoxide intermediate [R(H)O+ -O- ] that provides a key path in the chemistry which then follows. Formations of allylic hydroperoxides are unlikely from a 1 O2 'ene' reaction. Instead, the dihydrobenzofuran arises by 1 O2 oxidation facilitated by a 16° curvature of the glycocitrine ring imposed by a pyramidal N-methyl group. This curvature facilitates the formation of the iso-hydroperoxide, which is analogous to the iso species CH2 I+ -I- and CHI2 + -I- formed by UV photolysis of CH2 I2 and CHI3 . The iso-hydroperoxide is also structurally reminiscent of carbonyl oxides (R2 C=O+ -O- ) formed in the reaction of carbenes and oxygen. Our DFT results point to intermolecular process, in which the iso-hydroperoxide's fate relates to O-transfer and H2 O dehydration reactions for new insight into the biosynthesis of dihydrobenzofuran natural products.

Tuning the 1O2 Oxidation of a Phenol at the Air/Solid Interface of a Nanoparticle: Hydrophobic Surface Increases Oxophilicity.

Although silica surfaces have been used in organic oxidations for the production of peroxides, studies of airborne singlet oxygen at interfaces are limited and have not found widespread advantages. Here, with prenyl phenol-coated silica and delivery of singlet oxygen (1O2) through the gas phase, we uncover significant selectivity for dihydrofuran formation over allylic hydroperoxide formation. The hydrophobic particle causes prenyl phenol to produce an iso-hydroperoxide intermediate with an internally protonated oxygen atom, which leads to dihydrofuran formation as well as O atom transfer. In contrast, hydrophilic particles cause prenyl phenol to produce allylic hydroperoxide, due to phenol OH hydrogen bonding with SiOH surface groups. Mechanistic insight is provided by air/nanoparticle interfaces coated with the prenyl phenol, in which product yield was 6-fold greater on the hydrophobic nanoparticles compared to the hydrophilic nanoparticles and total rate constants (ASI-kT) of 1O2 were 13-fold greater on the hydrophobic vs hydrophilic nanoparticles. A slope intersection method was also developed that uses the airborne 1O2 lifetime (τairborne) and surface-associated 1O2 lifetime (τsurf) to quantitate 1O2 transitioning from volatile to non-volatile and surface boundary (surface···1O2). Further mechanistic insights on the selectivity of the reaction of prenyl phenol with 1O2 was provided by density functional theory calculations.

Singlet Oxygen Flux, Associated Lipid Photooxidation, and Membrane Expansion Dynamics Visualized on Giant Unilamellar Vesicles.

The physical properties of lipid membranes depend on their lipid composition. Photosensitized singlet oxygen (1O2) provides a handle to spatiotemporally control the generation of lipid hydroperoxides via the ene reaction, enabling fundamental studies on membrane dynamics in response to chemical composition changes. Critical to relating the physical properties of the lipid membrane to hydroperoxide formation is the availability of a sensitive reporter to quantify the arrival of 1O2. Here, we show that a fluorogenic α-tocopherol analogue, H4BPMHC, undergoes a >360-fold emission intensity enhancement in liposomes following a reaction with 1O2. Rapid quenching of 1O2 by the probe (kq = 4.9 × 108 M-1 s-1) ensures zero-order kinetics of probe consumption. The remarkable intensity enhancement of H4BPMHC upon 1O2 trapping, its linear temporal behavior, and its protective role in outcompeting membrane damage provide a sensitive and reliable method to quantify the 1O2 flux on lipid membranes. Armed with this probe, fluorescence microscopy studies were devised to enable (i) monitoring the flux of photosensitized 1O2 into giant unilamellar vesicles (GUVs), (ii) establishing the onset of the ene reaction with the double bonds of monounsaturated lipids, and (iii) visualizing the ensuing collective membrane expansion dynamics associated with molecular changes in the lipid structure upon hydroperoxide formation. A correlation was observed between the time for antioxidant H4BPMHC consumption by 1O2 and the onset of membrane fluctuations and surface expansion. Together, our imaging studies with H4BPMHC in GUVs provide a methodology to explore the intimate relationship between photosensitizer activity, chemical insult, membrane morphology, and its collective dynamics.

Diketopyrrolopyrrole Derivatives as Photosensitizing Agents against Staphylococcus aureus.

Diketopyrrolopyrrole (DPP) derivatives containing sulfonamide (Sulfonamide-DPP), pyridyl (Dipyridyl-DPP) and N-methylpyridyl (MePyridyl-DPP) substituents were assessed as antibacterial photosensitizers. Non-charged DPPs showed an intense absorption band centered at about 480 nm and green fluorescence emission (ΦF ~ 0.7) in acetonitrile. The absorption of MePyridyl-DPP was bathochromically shifted at 510 nm, with decreased fluorescence emission. Sulfonamide-DPP and Dipyridyl-DPP photosensitized the formation of O2 (1 Δg ) (ΦΔ ~ 0.15-0.17), while the production induced by MePyridyl-DPP was at least 10 times lower. Furthermore, these DPPs produced a photoreduction of NBT similar to that of the control. Photodynamic inactivation induced by DPPs was first investigated at the single-bacterium level of Staphylococcus aureus attached to a surface. After 30 min irradiation, MePyridyl-DPP produced a complete eradication of the bacteria. In bacterial cell suspensions, dicationic DPP induced more than 7 log10 decrease in S. aureus cell survival after 30 min irradiation. Potentiation with iodide anions allowed a complete elimination of bacteria after 15 min therapy. This compound was also effective to eliminate S. aureus cells on biofilms. The results show that MePyridyl-DPP bearing two positive groups provides an amphiphilic character to the structure that improves the interaction with the cell envelop. This effect enhances the photocytotoxic activity of MePyridyl-DPP against bacteria.

Sensitized Photooxidation of Ortho-Prenyl Phenol: Biomimetic Dihydrobenzofuran Synthesis and Total 1O2 Quenching.

The sensitized photooxidation of ortho-prenyl phenol is described with evidence that solvent aproticity favors the formation of a dihydrobenzofuran [2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran], a moiety commonly found in natural products. Benzene solvent increased the total quenching rate constant (kT ) of singlet oxygen with prenyl phenol by ~10-fold compared to methanol. A mechanism is proposed with preferential addition of singlet oxygen to prenyl site due to hydrogen bonding with the phenol OH group, which causes a divergence away from the singlet oxygen 'ene' reaction toward the dihydrobenzofuran as the major product. The reaction is a mixed photooxidized system since an epoxide arises by a type I sensitized photooxidation.

Porphyrin Polymers Bearing N,N'-Ethylene Crosslinkers as Photosensitizers against Bacteria.

The appearance of microbes resistant to antibiotics requires the development of alternative therapies for the treatment of infectious diseases. In this work two polymers, PTPPF16-EDA and PZnTPPF16-EDA, were synthesized by the nucleophilic aromatic substitution of 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin and its Zn(II) complex with ethylenediamine, respectively. In these structures, the tetrapyrrolic macrocycles were N,N'-ethylene crosslinked, which gives them greater mobility. The absorption spectra of the polymers showed a bathochromic shift of the Soret band of ~10 nm with respect to the monomers. This effect was also found in the red fluorescence emission peaks. Furthermore, both polymeric materials produced singlet molecular oxygen with high quantum yields. In addition, they were capable of generating superoxide anion radicals. Photodynamic inactivation sensitized by these polymers was tested in Staphylococcus aureus and Escherichia coli bacteria. A decrease in cell viability greater than 7 log (99.9999%) was observed in S. aureus incubated with 0.5 µM photosensitizer upon 30 min of irradiation. Under these conditions, a low inactivation of E. coli (0.5 log) was found. However, when the cells were treated with KI, the elimination of the Gram-negative bacteria was achieved. Therefore, these polymeric structures are interesting antimicrobial photosensitizing materials for the inactivation of pathogens.

Fluorescence-Amplified Detection of Redox Turnovers in Supported Lipid Bilayers Illuminates Redox Processes of α-Tocopherol.

Electron-transfer processes in lipid membranes are key to biological functions, yet challenging to study because of the intrinsic heterogeneity of the systems. Here, we report spectro-electrochemical measurements on indium tin oxide-supported lipid bilayers toward the selective induction and sensing of redox processes in membranes. Working at neutral pH with a fluorogenic α-tocopherol analogue, the dynamics of the two-electron oxidation of the chromanol to a chromanone and the rapid thermal decay of the latter to a chromoquinone are recorded as a rapid surge and drop in intensity, respectively. Continuous voltage cycling reveals rapid chromoquinone two-electron, two-proton reduction to dihydrochromoquinone at negative bias, followed by slow regeneration of the former at positive bias. The kinetic parameters of these different transitions are readily obtained as a function of applied potentials. The sensitivity and selectivity afforded by the reported method enables monitoring signals equivalent to femtoampere currents with a high signal-to-background ratio. The study provides a new method to monitor membrane redox processes with high sensitivity and minimal concentrations and unravels key dynamic aspects of α-tocopherol redox chemistry.

Antimicrobial Photosensitizing Material Based on Conjugated Zn(II) Porphyrins.

The widespread use of antibiotics has led to a considerable increase in the resistance of microorganisms to these agents. Consequently, it is imminent to establish new strategies to combat pathogens. An alternative involves the development of photoactive polymers that represent an interesting strategy to kill microbes and maintain aseptic surfaces. In this sense, a conjugated polymer (PZnTEP) based on Zn(II) 5,10,15,20-tetrakis-[4-(ethynyl)phenyl]porphyrin (ZnTEP) was obtained by the homocoupling reaction of terminal alkyne groups. PZnTEP exhibits a microporous structure with high surface areas allowing better interaction with bacteria. The UV-visible absorption spectra show the Soret and Q bands of PZnTEP red-shifted by about 18 nm compared to those of the monomer. Also, the conjugate presents the two red emission bands, characteristic of porphyrins. This polymer was able to produce singlet molecular oxygen and superoxide radical anion in the presence of NADH. Photocytotoxic activity sensitized by PZnTEP was investigated in bacterial suspensions. No viable Staphylococcus aureus cells were detected using 0.5 µM PZnTEP and 15 min irradiation. Under these conditions, complete photoinactivation of Escherichia coli was observed in the presence of 100 mM KI. Likewise, no survival was detected for E. coli incubated with 1.0 µM PZnTEP after 30 min irradiation. Furthermore, polylactic acid surfaces coated with PZnTEP were able to kill efficiently these bacteria. This surface can be reused for at least three photoinactivation cycles. Therefore, this conjugated photodynamic polymer is an interesting antimicrobial photoactive material for designing and developing self-sterilizing surfaces.

BOPHY-Fullerene C60 Dyad as a Photosensitizer for Antimicrobial Photodynamic Therapy.

A novel BOPHY-fullerene C60 dyad (BP-C60 ) was designed as a heavy-atom-free photosensitizer (PS) with potential uses in photodynamic treatment and reactive oxygen species (ROS)-mediated applications. BP-C60 consists of a BOPHY fluorophore covalently attached to a C60 moiety through a pyrrolidine ring. The BOPHY core works as a visible-light-harvesting antenna, while the fullerene C60 subunit elicits the photodynamic action. This fluorophore-fullerene cycloadduct, obtained by a straightforward synthetic route, was fully characterized and compared with its individual counterparts. The restricted rotation around the single bond connecting the BOPHY and pyrrolidine moieties led to the formation of two atropisomers. Spectroscopic, electrochemical, and computational studies disclose an efficient photoinduced energy/electron transfer process from BOPHY to fullerene C60 . Photodynamic studies indicate that BP-C60 produces ROS by both photomechanisms (type I and type II). Moreover, the dyad exhibits higher ROS production efficiency than its individual constitutional components. Preliminary screening of photodynamic inactivation on bacteria models (Staphylococcus aureus and Escherichia coli) demonstrated the ability of this dyad to be used as a heavy-atom-free PS. To the best of our knowledge, this is the first time that not only a BOPHY-fullerene C60 dyad is reported, but also that a BOPHY derivative is applied to photoinactivate microorganisms. This study lays the foundations for the development of new BOPHY-based PSs with plausible applications in the medical field.

Charge density distribution effect in pyrrolidine-fused chlorins on microbial uptake and antimicrobial photoinactivation of microbial pathogens.

Two novels structurally related pyrrolidine-fused chlorins were synthesized from 5,10,15,20-tetrakis(pentafluorophenyl)chlorin by nucleophilic aromatic substitution of the para-fluoro groups. The reaction with 2-dimethylaminoethanol produced TPCF16-NMe2 in 77% yield, while TPCF16-NBu was obtained using butylamine in 87% yield. The latter was extensively methylated to form TPCF16-N+Bu in 92% yield. The synthetic strategy was designed to compare the effect of charge density distribution on chlorin in the efficacy to induce photodynamic inactivation of pathogens. TPCF16-NMe2 has five tertiary amines that can acquire positive charges in aqueous medium by protonation. Furthermore, four of the cationic groups are located in amino groups linked to the chlorine macrocycle by an aliphatic structure of two carbon atoms, which gives it greater movement capacity. In contrast, TPCF16-N+Bu presents intrinsic positive charges on aromatic rings. Absorption and fluorescence emission properties were not affected by the peripheral substitution on the chlorin macrocycle. Both photosensitizers (PSs) were able to form singlet molecular oxygen and superoxide anion radical in solution. Uptake and photodynamic inactivation mediated by these chlorins were examined on Staphylococcus aureus and Escherichia coli. Both phototherapeutic agents produced efficient photoinactivation of S. aureus. However, only TPCF16-NMe2 was rapidly bound to E. coli cells and this chlorin was effective to photoinactivate both strains of bacteria using lower concentrations and shorter irradiation periods. Our outcomes reveal that the charge density distribution is a key factor to consider in the development of new PSs. Accordingly, this work stands out as a promising starting point for the design of new tetrapyrrolic macrocycles with application in PDI.

The Polarity of Fullerene C60 Derivatives for Enhanced Photodynamic Inactivation.

In this article, four novel fulleropyrrolidines derivatives were synthesized to study how the effect of polarity and positive charge distribution can influence the efficacy of photodynamic inactivation treatments to kill bacteria. The design of the photosensitizers was based on DFT calculations that allowed us to estimate the dipolar moment of the molecules. Neutral compounds bearing N-methyl bis-acetoxy-ethyl (1) and bis-hydroxyethyl (2) amine were the starting material to obtain the dicationic analogs N,N-dimethyl bis-methoxyethyl (3), and bis-acetoxy-ethyl) (4) methylammonio. As expected from fullerene C60 derivatives, compounds 1-4 absorb in the UV region, with a peak at 430 nm, a broader range of absorption up to 710 nm, and exhibit weak fluorescence emission in toluene and reverse micelles. In the biomimetic AOT micellar system, the highest singlet oxygen photosensitization was found for compounds 1, followed by 3, 2, and 4. Whereas 4 was the most effective reducing nitro blue tetrazolium in the presence of ß-NADH. The influence of type I and type II mechanism on the photodynamic activity of compounds 3 and 4 was further examined in the presence of L-tryptophan and two reactive oxygen species scavengers. In vitro experiments indicated that the compounds with the highest dipolar moments, 3 (37.19 D) and 4 (38.46 D), inactivated methicillin-resistant Staphylococcus aureus and Escherichia coli bacteria using an energy dose <2.4 J cm-2 . No inactivation was observed for the neutral analogs with the lowest dipolar moments. These findings help to optimize sensitizer structures to improve photodynamic inactivation.

Interparticle Delivery and Detection of Volatile Singlet Oxygen at Air/Solid Interfaces.

An interparticle system has been devised, allowing airborne singlet oxygen to transfer between particle surfaces. Singlet oxygen is photogenerated on a sensitizer particle, where it then travels through air to a second particle bearing an oxidizable compound-a particulate-based approach with some similarities to reactive oxygen quenching in the atmosphere. In atmospheric photochemistry, singlet oxygen is generated by natural particulate matter, but its formation and quenching between particles has until now not been determined. Determining how singlet oxygen reacts on a second surface is useful and was developed by a three-phase system (particle-air-particle) interparticulate photoreaction with tunable quenching properties. We identify singlet oxygen quenching directly by near-IR phosphorescence in the airborne state and at the air/particle interface for total quenching rate constants (kT) of adsorbed anthracene trapping agents. The air/solid interface kT of singlet oxygen by anthracene-coated particles was (2.8 ± 0.8) × 107 g mol-1 s-1 for 9,10-dimethylanthracene and (2.1 ± 0.9) × 107 g mol-1 s-1 for 9,10-anthracene dipropionate dianion, and the lifetime of airborne singlet oxygen was measured to be 550 µs. These real-time interactions and particle-induced quenching steps open up new opportunities for singlet oxygen research of atmospheric and particulate processes.

Self-Sterilizing 3D-Printed Polylactic Acid Surfaces Coated with a BODIPY Photosensitizer.

Herein, we report the use of polylactic acid coated with a halogenated BODIPY photosensitizer (PS) as a novel self-sterilizing, low-cost, and eco-friendly material activated with visible light. In this article, polymeric surfaces were 3D-printed and treated with the PS using three simple methodologies: spin coating, aerosolization, and brush dispersion. Our studies showed that the polymeric matrix remains unaffected upon addition of the PS, as observed by dynamic mechanical analysis, Fourier transform infrared, scanning electron microscopy (SEM), and fluorescence microscopy. Furthermore, the photophysical and photodynamic properties of the dye remained intact after being adsorbed on the polymer. This photoactive material can be reused and was successfully inactivating methicillin-resistant Staphylococcus aureus and Escherichia coli in planktonic media for at least three inactivation cycles after short-time light exposure. A real-time experiment using a fluorescence microscope showed how bacteria anchored to the antimicrobial surface were inactivated within 30 min using visible light and low energy. Moreover, the material effectively eradicated these two bacterial strains on the first stage of biofilm formation, as elucidated by SEM. Unlike other antimicrobial approaches that implement a dissolved PS or non-sustainable materials, we offer an accessible green and economic alternative to acquire self-sterilizing surfaces with any desired shape.

Revealing ROS Production by Antibiotics and Photosensitizers in Biofilms: A Fluorescence Microscopy Approach.

Reactive oxygen species (ROS) production within biofilms is studied with a simple and easy setup based on fluorescence microscopy. Herein, a biofilm is exposed to different ROS inducers: a bactericidal antibiotic (ciprofloxacin) and a BODIPY-based photosensitizer (I2B-OAc). Real-time ROS induction in the core of the biofilms is monitored utilizing two fluorescent reporters-AMDA and H2DCFDA-the first one with selectivity toward singlet oxygen (1O2) and the latest for other ROS (O2•-, H2O2, and OH•-). A point-by-point methodology is reported, starting with the sample preparation all the way through the microscope setup and, finally, processing of the images.

Potentiation Effect of Iodine Species on the Antimicrobial Capability of Surfaces Coated with Electroactive Phthalocyanines.

The spreading of different infections can occur through direct contact with glass surfaces in commonly used areas. Incorporating the use of alternative therapies in these materials seems essential to reduce and also avoid bacterial resistance. In this work, the capability to kill microbes of glass surfaces coated with two electroactive metalated phthalocyanines (ZnPc-EDOT and CuPc-EDOT) is assessed. The results show that both of these materials are capable of producing reactive oxygen species; however, the polymer with Zn(II) (ZnPc-PEDOT) has a singlet oxygen quantum yield 8-fold higher than that of the Cu(II) containing analogue. This was reflected in the in vitro experiments where the effectiveness of the surfaces was tested in bacterial suspensions, monitoring single microbe inactivation upon attachment to the polymers, and eliminating mature biofilms. Furthermore, we evaluated the use of an inorganic salt (KI) to potentiate the photodynamic inactivation mediated by an electropolymerized surface. The addition of the salt improved the efficiency of phototherapy at least two times for both polymers; nevertheless, the material coated with ZnPc-PEDOT was the only one capable of eliminating >99.98% of the initial microbes loading under different circumstances.

Photoactive antimicrobial coating based on a PEDOT-fullerene C60 polymeric dyad.

A photostable and photodynamic antimicrobial surface was successfully obtained and applied to photoinactivate microorganisms. This approach was based on the synthesis of a fullerene C60 derivative (EDOT-C60) where fullerene C60 is covalently linked to 3,4-ethylenedioxythiophene (EDOT) through a 1,3-dipolar cycloaddition reaction. This dual-functional monomer bears an EDOT center connected via an alkyl chain to a fullerene C60 moiety. In this structure, EDOT acts as an electropolymerizable unit that allows the film formation over conducting substrates, while fullerene C60 performs the photodynamic antimicrobial activity. Electrochemical polymerization of EDOT was used to obtain stable and photodynamic polymeric films (PEDOT-C60) in a controllable procedure. Cyclic voltammetry and UV-visible spectroscopy studies showed that the fullerene C60 units were not altered during the electropolymerization process, obtaining surfaces with high fullerene content. Photobleaching measurements demonstrated that the electropolymerized films were highly photostable. Moreover, photodynamic properties of PEDOT-C60 were compared with fullerene C60 and showed that electrodeposited films were able to generate reactive oxygen species (ROS) through the two photomechanisms, producing singlet molecular oxygen (type II) and superoxide radical anion (type I). All studies demonstrated that fullerene C60 moieties covalently attached to the polymeric matrix mainly conserve the photodynamic characteristics. Hence, photodynamic action sensitized by PEDOT-C60 was assessed in vitro against Staphylococcus aureus. The photosensitized inactivation by the electropolymerized films on bacteria suspensions produced >99.9% reduction in S. aureus survival. Fluorescence microscopy experiments with S. aureus adhered to the PEDOT-C60 surface showed a complete microbe annihilation. Also, the eradication of biofilms formed on PEDOT-C60 surfaces resulted in a photokilling >99.9% after visible light irradiation. Our results demonstrated that these antimicrobial photodynamic polymeric films are a promising and versatile platform to photoinactivate microorganisms and to obtain photostable self-sterilizing surfaces.

BODIPYs bearing a dimethylaminopropoxy substituent for imaging and photodynamic inactivation of bacteria.

A new BODIPY (BDP 1) bearing a dimethylaminopropoxy group attached to a phenylene unit was synthesized. This compound was brominated to obtain the halogenated analog BDP 2, which was designed to enhance the photodynamic effect of BODIPY to kill bacteria without an intrinsic cationic charge. The basic amino group located at the end of the propoxy bridge can acquire a positive charge by protonation in an aqueous medium, increasing the binding to bacterial cells. Interaction and photokilling activity mediated by these compounds was evaluated in Staphylococcus aureus and Escherichia coli. BDP 1 and BDP 2 were rapidly bound to bacterial cells, showing bioimages with green emission. Complete elimination of S. aureus was detected when cells were incubated with 1 µM BDP 2 and irradiated for 5 min. Comparable photoinactivation was obtained with E. coli, after an irradiation of 30 min. Furthermore, BDP 2 was effective to kill bacteria at very low concentration (0.5 µM). Thus, BDP 1 showed mainly interesting properties as a fluorophore, whereas BDP 2 was highly effective photosensitizer as a broad-spectrum antibacterial agent.

Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic.

The formation of reactive oxygen species (ROS) induced by bactericidal antibiotics has been associated with a common, nonspecific mechanism of cellular death. Herein, we report real-time single-cell fluorescence studies on Escherichia coli stained with a fluorogenic probe for lipid peroxyl radicals showing the generation of this form of ROS when exposed to the minimum inhibitory concentration (MIC) and 10× MIC of the fluoroquinolone antibiotic ciprofloxacin (3 and 30 µM, respectively). Single-cell intensity-time trajectories show an induction period followed by an accelerating phase for cells treated with antibiotic, where initial and maximum intensity achieved following 3.5 h of incubation with antibiotic showed dose-dependent average values. A large fraction of bacteria remains viable after the studies, indicating ROS formation is occurring a priori of cell death. Punctate structures are observed, consistent with membrane blebbing. The addition of a membrane embedding lipid peroxyl radical scavenger, an α-tocopherol analogue, to the media increased the MIC of ciprofloxacin. Lipid peroxyl radical formation precedes E. coli cell death and may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Altogether, our work illustrates that lipid peroxidation is caused by ciprofloxacin in E. coli and suppressed by α-tocopherol analogues. Lipid peroxidation may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Our work provides a methodology to assess antibiotic-induced membrane peroxidation at the single-cell level; this methodology provides opportunities to explore the scope and nature of lipid peroxidation in antibiotic-induced cell lethality.

Magnetic Nanoplatforms for in Situ Modification of Macromolecules: Synthesis, Characterization, and Photoinactivating Power of Cationic Nanoiman-Porphyrin Conjugates.

A nanoplatform concept was developed to synthesize accessible photoactive magnetic nanoparticles (MNPs) of Fe3O4 coated with silica. This approach was based on the covalent binding of 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPPF20) to aminopropyl-grafted MNPs by nucleophilic aromatic substitution reaction (SNAr) to obtain conjugate MNP-P1. After in situ modification, the remaining pentafluorophenyl groups of TPPF20 attached to MNPs were substituted by dimethylaminoethoxy groups to form MNP-P2. The basic amine group of these conjugates can be protonated in aqueous media. In addition, MNP-P1 and MNP-P2 were intrinsically charged to produce cationic conjugates MNP+-P1 and MNP+-P2+ by methylation. All of them were easily purified by magnetic decantation in high yields. The average size of the MNPs was ∼15 nm, and the main difference between these conjugates was the greater coating with positive charges of MNP+-P2+, as shown by the zeta potential values. Absorption spectra exhibited the Soret and Q bands characteristic of TPPF20 linked to MNPs. Furthermore, these conjugates showed red fluorescence emission of porphyrin with quantum yields of 0.011-0.036. The photodynamic effect sensitized by the conjugates indicated the efficient formation of singlet molecular oxygen in different media, reaching quantum yield values of 0.17-0.34 in N,N-dimethylformamide. The photodynamic activity of the conjugates was evaluated to inactivate the Gram-positive bacteria Staphylococcus aureus, the Gram-negative bacteria Escherichia coli, and the yeast Candida albicans. The modified cationic MNP+-P2+ was the most effective conjugate for photodynamic inactivation (PDI) of microorganisms. Binding of this conjugate to bacteria and photoinactivation capability was checked by means of fluorescence microscopy. Also, sustainable use by recycling was determined after three PDI treatments. Therefore, this methodology is a suitable scaffold for the in situ modification of conjugates, and in particular, MNP+-P2+ represents a useful photodynamic active material to eradicate microorganisms.