Photochemistry and Photobiology

In antimicrobial photodynamic therapy, the photosensitizer (PS) in its ground singlet state absorbs light to give the excited singlet state that can transition to the long-lived triplet state. This PS triplet may undergo energy transfer (Type 2) or electron transfer (Type 1) to oxygen to form reactive oxygen species (singlet oxygen and/or hydroxyl radicals) that can kill both Gram-positive and Gram-negative bacteria and fungi. Infections in animal models can also be treated.

In just 20 years antimicrobial photodynamic therapy (PDT) has emerged as a discovery and development platform inspiring a proliferation of light-based antimicrobial explorations worldwide. However, the potential for microbial resistance development using PDT remains under-investigated. Studies of resistance have been sporadic but they are rapidly increasing, with recent reports examining key elements of the microbial phenotype. These include multidrug efflux systems, biofilm, spore formation, virulence and pathogenicity determinants. The emerging consensus is that the effectiveness of PDT may be profoundly impacted by all these systems, but the exact mechanisms of these effects remain elusive. For example, an array of studies suggests that antimicrobial photosensitizers are substrates of multidrug efflux systems. An evolving antimicrobial discovery concept is based on the exploration of synergies between photosensitizers and small molecules efflux pump inhibitors. PS photosensitizer (red), EPI efflux pump inhibitor (green).

Superficial cutaneous mycoses are probably the most prevalent infectious diseases worldwide, caused by dermatophytes or opportunistic yeasts. Up to now the management of fungi infections has been based on the use of oral and topical antifungal agents, however, main limitations of the current therapeutic antifungal options include recurrences of the infection, need for prolonged treatments, side effects, drug interactions and excessive costs. Antimicrobial photodynamic therapy (PDT) could represent a suitable alternative for the treatment of localized fungal infections. We review the studies published to date on antifungal applications of PDT, with special focus on Candida albicans (Candida spores in the image).

New derivatives of the standard phenothiazinium photosensitizers methylene blue and toluidine blue exhibited much improved activity, compared with the lead compounds, against Propionibacterium acnes, the main bacterium associated with acne vulgaris. The new derivatives were also photobactericidal at much lower concentrations than the topical agent benzoyl peroxide and standard tetracyclines. Bactericidal activity was also significantly more rapid than the tetracyclines.

To combat the escalating threat to human health posed by the transmission of pathogenic bacteria that adhere to surfaces, we investigated the photobactericidal activity of cellulose nanocrystals that have been modified with a porphyrin-derived photosensitizer. These porphyrin-cellulose nanocrystals exhibited broad spectrum antimicrobial activity, with the best results achieving an impressive 5–6 log units reduction in colony forming units (99.999 + % killing) for Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii (MDRAB), and methicillin-resistant Staphylococcus aureus (MRSA), and a noteworthy 99.9 + % sterilization of Pseudomonas aeruginosa.

Three new cationic thio-pyridinium phthalocyanines (3a–5a) were prepared and their potential as photosensitizers (PSs) was determined against Escherichia coli. Derivative 3a was the most effective PS, causing a 5 logs reduction in bioluminescence under white or red lights.

The surface of temoporfin-loaded liposomes was modified with a bacteria-targeting ligand, wheat germ agglutinin, to prepare bacteria-targeting liposomes, which bind to bacteria and consequently increase the delivery of temoporfin to bacteria. After light illumination, the photosensitizers (temoporfin) generate reactive oxygen species, resulting in photodynamic inactivation of bacteria.

Among Gram-negative bacteria, Pseudomonas aeruginosa is one of the least susceptible to photodynamic inactivation. A tetracationic meso-arylsubstituted porphyrin (RM24) was used to photoeradicate P. aeruginosa PAO1. The photo-oxidative stress induced by RM24 was dependent on the light dose and was strictly correlated to the amount of photosensitizer bound to the cells, whereas the uptake was inversely correlated to the amount of organic matter in the medium. RM24 was able to generate singlet oxygen; however, reactive oxygen species formation cannot be ruled out.

Condylomata acuminata (CA) are the most prevalent sexually transmitted disease and closely associated with human papillomavirus (HPV) infections. Although CA mainly occur in the external genital and perianal area, HPV infection in these areas in women can lead to further infections in the vaginal and cervical mucosal epithelia. This clinical study investigated the efficacy of ALA PDT in treatment of HPV-associated cervical condylomata. We demonstrated that topical ALA PDT could effectively eradicate cervical HPV infection and ablate warty lesion (illustrated).

Erythrosine (ER)-mediated photodynamic inactivation (PDI) was able to kill microorganisms. Chitosan nanoparticles loaded with ER exhibited better PDI efficacy than ER alone on microbial cultures and biofilms.

The present study highlighted the importance of the inherent cell membrane permeabilizing effect of chitosan and increased cell/biofilm uptake of conjugated photosensitizer (CSRB) to produce significant antibiofilm efficacy during photodynamic therapy. CSRB was found to be significantly better in eliminating both gram-positive and gram-negative bacterial biofilms as compared to rose bengal and methylene blue.

This study reports a potential use of antimicrobial photodynamic therapy (aPDT) to arrest caries progression. We developed a rat model of incipient caries to investigate the short-term effects of aPDT on oral microbiota regulation and enamel demineralization. Induced caries in rats’ molars was confirmed by optical coherence tomography (OCT) before aPDT. Bacterial load following aPDT remained significantly lower than untreated group until 10 days post-treatment. Data obtained from OCT signal showed that aPDT group presented lesser mineral loss corroborating caries arrestment. Put together, these findings suggest that aPDT could be a new approach for public health.

This study reports a potential use of antimicrobial photodynamic therapy (aPDT) to delay bacteremia and increase life expectancy. We developed a mouse model of P. aeruginosa-infected burns to investigate the effects of aPDT in situ and in the bloodstream. Infected mice without treatment died in 18 h. APDT was able to reduce bacterial load in burn wounds, retard bacteremia and keep the bacterial levels in blood 2–3 logs lower compared with untreated group. Mice survival was increased by 24 h. Put together, these findings suggest that aPDT could be a new prophylactic approach in the care of burned patients.

The confocal image shows yeast cells of the fungus Candida albicans stained with MitoTracker Red (Molecular Probes, Eugene, OR), visualizing ribbon-like mitochondria. Inhibition of mitochondrial respiration in Candida increases the efficacy of photodynamic therapy (PDT). In addition, the antifungal drug miconazole has adverse effects on C. albicans mitochondrial function. Our recent work demonstrates that efficacy of PDT against C. albicans is enhanced in combination with miconazole. Combined PDT-miconazole treatment may lower the effective dose of PDT against Candida, as well as reducing the frequency of application for an antifungal agent widely used for treatment of cutaneous and mucosal candidiasis.

The mechanism of photoinactivation of Candida albicans by uncharged, cationic or anionic porphyrins under blue light, was found to be dependent on the uptake of the porphyrins into yeast cells, and was also dependent on the protein’s quantity and quality in the photosensitization medium. Only phototreatment in phosphate buffered saline resulted in eradication with all three porphyrins. X-ray microanalysis demonstrated that only with the uncharged or cationic porphyrins in a protein-poor medium exhibited ion loss, indicating cell-membrane damage. Transmission electron microscopy indicated cellular and chromosomal damage. Only taken up porphyrins might lead (upon illumination) to processes that facilitate the formation of reactive oxygen species that will damage and inactivate the yeast cells.

Photodynamic treatments using hypericin show a fungicidal effect on Candida spp., the most sensitive strain being Candida albicans and the most resistant Candida krusei. C. albicans infections could be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.

Leishmania parasitize the phagolysosomes of antigen-presenting cells and are thus uniquely suitable for targeted delivery of vaccines. When preloaded exogenously with photosensitizers, e.g. aluminum phthalocyanine chloride (AlPhCl; A) or when exposed to aminolevulinate as transgenic mutants to accumulate uroporphyrin endogenously (B), Leishmania are photolyzed in these cells selectively, but incompletely. Double photosensitization of Leishmania with both (C) increases their photolysis significantly so that no survivors are detectable in both in vitro and in vivo systems. The potential utility of Leishmania as a photodynamic vaccine carrier is thus enhanced by the double photosensitization to provide a favorable profile of their safety.

Increasing prevalence of antibiotic-resistant bacteria is one of the most pressing global health issues. It is imperative to develop nonantibiotic approaches for combating antibiotic-resistant bacterial infections. Photodynamic antimicrobial chemotherapy (PACT) is a promising alternative. This study examined the effect of hypericin-mediated PACT on clinically isolated S. aureus and E. coli. Hypericin-mediated PACT could induce significant killing of Gram positive methicillin-sensitive and methicillin-resistant S. aureus cells (illustrated), but was ineffective on Gram negative E. coli cells. This phenomenon is due to the differences in bacterial wall/membrane structures that directly affect cellular uptake of hypericin.

Upon light irradiation, the 1,3-diarylisobenzofurans undergo a charge transfer on the initially planar structure, followed by relaxation to an intramolecular twisted excited state responsible for the intense emission of such compounds. Experimental and theoretical results evidence this process.

Ciprofloxacin triplet state (³CPX^*) can be quenched by TMPD, FCA and dGMP, with rate constants of 1.8 × 10⁹, 1.5 × 10⁹ and 5.8 × 10⁷ dm³ mol^-1 s^-1, respectively. The formation rates of CPX radical anion (CPX^·−) was determined to be 1.5 × 10⁹ dm³ mol^-1 s^-1. The triplet energy of CPX was determined to be 262 kJ mol⁻¹. The scheme of electron transfer from TMPD, FCA and dGMP to ³CPX^* was proposed. The direct time-resolved spectroscopic evidence obtained will be helpful in elucidating the mechanism involved in FQs-photoinduced DNA damage.

Early, easy chemically induced DNA damage detection may help prevent disease and aid cancer treatment. Molecular beacon probes can be used for the detection of chemically induced DNA damage due to their sensitivity and specificity. This article highlights the ability of molecular beacons to detect DNA damage induced by psoralen, a phototherapeutic drug that upon exposure to UVA light forms adducts with both ssDNA and dsDNA. The molecular beacons provide a rapid, mix-and-read assay that can discriminate between undamaged and damaged ssDNA without the need to isolate the damaged ssDNA from the solution nor from the damage agent.

Chlorophyll a and b differ solely in the nature of the 7-substituent, whereas chlorophyll a and d differ solely in the 3-substituent, yet have distinct long-wavelength absorption maxima: 665 (a) 646 (b) and 692 nm (d). Herein, the spectra, singlet excited-state decay characteristics, and results from DFT calculations are examined for synthetic chlorins and 13¹-oxophorbines that contain a variety of substituents at the 3-, 7- and/or 13-positions. An intricate interplay of site-specific electronic effects of substituents on the four frontier MOs of the chlorin macrocycle is found to govern the spectral properties.

Exceedingly efficient intrachlorosomal excitation energy transfer channel (sub 300 fs) in chlorosomes from photosynthetic green sulfur bacteria Chlorobaculum (Cba.) tepidum and Prosthecochloris (Pst.) aestuarii was identified by time-resolved femtosecond transient absorption spectroscopy. Anisotropy decay results suggested characteristic excitation transfer times from the red-most chlorosome antenna elements to the baseplate of 6.6, 8.8 ps and 12.1 ps for chlorosomes from Chloroflexus aurantiacus, Pst. aestuarii and Cba. tepidum, respectively. One picosecond anisotropy component, common to all three species, was assigned to energy transfer between bacteriochlorophyll a molecules of the baseplate.

Microalgae alter their cellular lipid content depending on the environmental conditions. The unicellular planktonic marine species, Nannochloropsis spp., increase cellular lipid content when under stress. In this study, we examined the individual and combined effects of UV-A exposure and nutrient limitation on lipid accumulation in Nannochloropsis oculata. UV-A increased cellular lipid content of N. oculata, and the increase was directly proportional to the UV-A dose employed. Cellular lipid also increased with a decrease in nutrient concentration. Enhanced UV-A and nutrient limitation together had a synergistic effect, resulting in an increase in cellular lipids of N. oculata.

Haloquadratum walsbyi is a peculiar organism which often dominates the microbial communities of the hypersaline ecosystems, such as salt lakes and solar saltern crystallizer ponds. It is extremely thin and possesses a unique square-like shape, with sharp edges and acute straight corners. The flat cells form large sheets similar to solar panels, able to efficiently collect light as an energy source for metabolism. Like other archaeal extremely halophilic microorganisms, square cells encode light-activated retinal-proteins to survive in hypersaline environments. In this study we describe the biochemical properties and the photochemistry of the light-activated proton pump Bop I of H. walsbyi, grown in laboratory.

UV exposure is known to cause morphological deformities in fish embryos but the molecular mechanisms are unclear. Gene expression of osteonectin and p53 increases during UV exposure of zebrafish embryos. Expression is mainly a function of UVB exposure. Microinjection of osteonectin mRNA causes similar morphological deformities as UV exposure. This advances our understanding of the molecular basis of UV effects and importance of osteonectin in embryonic development.

UV-irradiated cells transmit signals to nonirradiated cells through secretory factors (UVRF) that can produce a variety of responses. We have found that cells exposed to these factors were less sensitive to killing by UVC or H₂O₂, but not to MNNG. For UVC and H₂O₂ treatment oxidative damage was lowered because UVRF exposed cells have elevated CAT and SOD activities. The ability of these cells to counter the assaults by oxidants through a considerable increase in GPx activity is significant for its ability to provide protection to cells.

The anthracene derivative termed diethyl-3-3′-(9,10-anthracenediyl)bis acrylate (DADB) is converted to an endoperoxide by the action of singlet oxygen, resulting in the loss of fluorescence. This figure illustrates the utilization of DADB to assess formation of singlet oxygen in cell culture during photodynamic therapy. Left: control cells; right: after an LD90 PDT dose using the chlorin (NPe6).

In this study, we evaluated the effect of photodynamic therapy in a highly metastatic human osteosarcoma cell line after application of Foslipos^®, a liposomal formulation of 5,1,15,20-tetrakis(meta-hydroxyphenyl)chlorine (mTHPC). Confocal Microscopy displaying Foslipos uptake of 143B cells after 5 h incubation with 2.5 μg mL⁻¹ of the photosensitizer is visible in the figure: images in the three panels represent DAPI (left panel), Foslipos (middle panel) and the overlay image of the previous two (right panel). Nuclear localization is not observable.

Photographs of Enterobacter coli colonies on LB agar incubated for 18 h at 37°C. Plates represent E. coli cells within a 100 μL distilled water suspension after photocatalytic treatment with Degussa P25 and UVA irradiation for (A) 0 min; (B) 120 min; (C) 240 min; and (D) 360 min. As treatment progresses the diameter of a typical colony is significantly reduced from 15 mm, until at 360 min, colonies are so small (3 mm) that it is difficult to distinguish them with the naked eye. Small colony variants are thought to occur due to sublethal ^•OH attack.

Measurements were conducted at San Ya, China (18.4°N, 109.7°E, altitude 18 m) to investigate the diurnal variation of ocular exposure to ultraviolet radiation, which could simulate three different types of exposure (maximum, average and minimum) by rotating the manikin. The diurnal variations of the ocular absolute irradiance and biologically effective UV irradiances in the maximum exposure were both different from the results of the ambient UV measurements. The results showed that protection against ocular overexposure during outdoor activities should be taken not only at noon but also at other times.

For two decades manufacturers have sought to develop sunscreens with spectral profiles that approach uniform absorption across the solar ultraviolet spectrum. There has been considerable success in this regard and today we have available products that virtually meet this criterion of spectral uniformity. Yet to ensure optimal compliance by consumers, sunscreens need to be colorless when applied to the skin and hence their absorbance needs to fall to zero as the nonvisible UV wavelengths merge into the visible region. This figure compares our proposed ideal spectral profile of a topical sunscreen with the spectral sensitivity of the eye.

Formulations containing, or not, 10% of a dispersion of liposome with magnesium ascorbyl phosphate (MAP), alpha-lipoic acid (ALA) and kinetin were evaluated. The in vitro studies showed that this association in liposomes possesses free-radical scavenging properties. The in vivo studies on animal model showed that this association protected skin barrier function against UV damage, seen by transepidermal water loss reduction. Clinical studies performed on human volunteers, forearms showed that the formulation with the active substances enhanced skin moisture and altered skin viscoelastic properties when compared with the vehicle, which suggests that kinetin, MAP and ALA in liposomes had sustained hydration effect and acted in the deeper layers of the skin provoking alterations in the viscoelastic-to-elastic ratio.

Most recently, light-controlled drug release systems were proposed, where singlet oxygen generated from low energy light and a photosensitizer cleaves electron-rich alkenes, e.g. 1,2-diaryloxyethene. However, the synthesis of 1,2-diaryloxyethene was not well established. Herein, we report a facile synthesis of 1,2-diaryloxyethenes and its sulfur and nitrogen analogs. Model compounds were prepared in four steps from 4-phenylphenol via Ullman-type coupling reaction.