Synthesis and properties of benzo[a]phenoxazinium chalcogen analogues as novel broad-spectrum antimicrobial photosensitizers.

The goal of this investigation was to develop improved photosensitizers for use as antimicrobial drugs in photodynamic therapy of localized infections. Replacement of the oxygen atom in 5-(ethylamino)-9-diethylaminobenzo[a]phenoxazinium chloride (1) with sulfur and selenium afforded thiazinium and selenazinium analogues 2 and 3, respectively. All three dyes are water soluble, lipophilic, and red light absorbers. The relative photodynamic activities of the chalcogen series were evaluated against a panel of prototypical pathogenic microorganisms: the Gram-positive Enterococcus faecalis, the Gram-negative Escherichia coli, and the fungus Candida albicans. Selenium dye 3 was highly effective as a broad-spectrum antimicrobial photosensitizer with fluences of 4-32 J/cm2 killing 2-5 more logs of all cell types than sulfur dye 2, which was slightly more effective than oxygen analogue 1. These data, taken with the findings of uptake and retention studies, suggest that the superior activity of selenium derivative 3 can be attributed to its much higher triplet quantum yield.

Use of chitosan bandage to prevent fatal infections developing from highly contaminated wounds in mice.

HemCon bandage is an engineered chitosan acetate preparation used as a hemostatic control dressing, and its chemical structure suggests that it should also be antimicrobial. We tested its ability to rapidly kill bacteria in vitro and in mouse models of infected wounds. We used the Gram-negative species Pseudomonas aeruginosa and Proteus mirabilis and the Gram-positive Staphylococcus aureus that had all been stably transduced with the entire bacterial lux operon to allow in vivo bioluminescence imaging. An excisional wound in Balb/c mice was inoculated with 50-250 million cells followed after 30 min by application of HemCon bandage, alginate sponge bandage, silver sulfadiazine cream or no treatment. HemCon was more adhesive to the wound and conformed well to the injury compared to alginate. Animal survival was followed over 15 days with observations of bioluminescence emission and animal activity daily. Chitosan acetate treated mice infected with P. aeruginosa and P. mirabilis all survived while those receiving no treatment, alginate and silver sulfadiazine demonstrated 25-100% mortality. Chitosan acetate was much more effective than other treatments in rapidly reducing bioluminescence in the wound consistent with its rapid bactericidal activity in vitro as well as its light-scattering properties. S. aureus formed only non-lethal localized infections after temporary immunosuppression of the mice but HemCon was again more effective in reducing bioluminescence. The data suggest that chitosan acetate rapidly kills bacteria in the wound before systemic invasion can take place, and is superior to alginate bandage and silver sulfadiazine that may both encourage bacterial growth in the short term.

Protease-stable polycationic photosensitizer conjugates between polyethyleneimine and chlorin(e6) for broad-spectrum antimicrobial photoinactivation.

We previously showed that covalent conjugates between poly-L-lysine and chlorin(e6) were efficient photosensitizers (PS) of both gram-positive and gram-negative bacteria. The polycationic molecular constructs increased binding and penetration of the PS into impermeable gram-negative cells. We have now prepared a novel set of second-generation polycationic conjugates between chlorin(e6) and three molecular forms of polyethyleneimine (PEI): a small linear, a small cross-linked, and a large cross-linked molecule. The conjugates were characterized by high-pressure liquid chromatography and tested for their ability to kill a panel of pathogenic microorganisms, the gram-positive Staphylococcus aureus and Streptococcus pyogenes, the gram-negative Escherichia coli and Pseudomonas aeruginosa, and the yeast Candida albicans, after exposure to low levels of red light. The large cross-linked molecule efficiently killed all organisms, while the linear conjugate killed gram-positive bacteria and C. albicans. The small cross-linked conjugate was the least efficient antimicrobial PS and its remarkably low activity could not be explained by reduced photochemical quantum yield or reduced cellular uptake. In contrast to polylysine conjugates, the PEI conjugates were resistant to degradation by proteases such as trypsin that hydrolyze lysine-lysine peptide bonds, The advantage of protease stability combined with the ready availability of PEI suggests these molecules may be superior to polylysine-PS conjugates for photodynamic therapy of localized infections.

Photodynamic inactivation of Bacillus spores, mediated by phenothiazinium dyes.

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.

Cationic fullerenes are effective and selective antimicrobial photosensitizers.

Fullerenes are soccer ball-shaped molecules composed of carbon atoms, and, when derivatized with functional groups, they become soluble and can act as photosensitizers. Antimicrobial photodynamic therapy combines a nontoxic photosensitizer with harmless visible light to generate reactive oxygen species that kill microbial cells. We have compared the antimicrobial activity of six functionalized C(60) compounds with one, two, or three hydrophilic or cationic groups in combination with white light against gram-positive bacteria, gram-negative bacteria, and fungi. After a 10 min incubation, the bis- and tris-cationic fullerenes were highly active in killing all tested microbes (4-6 logs) under conditions in which mammalian cells were comparatively unharmed. These compounds performed significantly better than a widely used antimicrobial photosensitizer, toluidine blue O. The high selectivity and efficacy exhibited by these photosensitizers encourage further testing for antimicrobial applications.

Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria.

The increasing occurrence of multi-antibiotic resistant microbes has led to the search for alternative methods of killing pathogens and treating infections. Photodynamic therapy (PDT) uses the combination of non-toxic dyes and harmless visible light to produce reactive oxygen species that can kill mammalian and microbial cells. Although the photodynamic inactivation of bacteria has been known for over a hundred years, its use to treat infections has not been much developed. This may be partly due to the difficulty of monitoring the effectiveness of PDT in animal models of infection. In order to facilitate this monitoring process, we have developed a procedure that uses bioluminescent genetically engineered bacteria and a light sensitive imaging system to allow real-time visualization of infections. When these bacteria are treated with PDT in vitro, the loss of luminescence parallels the loss of colony-forming ability. We have developed several models of infections in wounds and soft-tissue abscesses in mice that can be followed by bioluminescence imaging. The size and intensity of the infection can be sequentially monitored in a non-invasive fashion in individual mice in real-time. When photosensitizers are introduced into the infected tissue followed by illumination with red light, a light-dose dependent loss of luminescence is seen. If the bacterium is invasive, the loss of luminescence correlates with increased survival of the mice, whilst animals in control groups die of sepsis within five days. Healing of the PDT treated wounds is not impaired and may actually be improved. This approach can allow many animal models of localized infections to be accurately monitored for efficacy of treatment by PDT.

Photodynamic therapy for Staphylococcus aureus infected burn wounds in mice.

The rise of multiply antibiotic resistant bacteria has led to searches for novel antimicrobial therapies to treat infections. Photodynamic therapy (PDT) is a potential candidate; it uses the combination of a photosensitizer with visible light to produce reactive oxygen species that lead to cell death. We used PDT mediated by meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin (PTMPP) to treat burn wounds in mice with established Staphylococcus aureus infections The third degree burn wounds were infected with bioluminescent S. aureus. PDT was applied after one day of bacterial growth by adding a 25% DMSO/500 microM PTMPP solution to the wound followed by illumination with red light and periodic imaging of the mice using a sensitive camera to detect the bioluminescence. More than 98% of the bacteria were eradicated after a light dose of 210 J cm(-2) in the presence of PTMPP. However, bacterial re-growth was observed. Light alone or PDT both delayed the wound healing. These data suggest that PDT has the potential to rapidly reduce the bacterial load in infected burns. The treatment needs to be optimized to reduce wound damage and prevent recurrence.

Effect of cell-photosensitizer binding and cell density on microbial photoinactivation.

Photodynamic therapy involves the use of nontoxic dyes called photosensitizers and visible light to produce reactive oxygen species and cell killing. It is being studied as an alternative method of killing pathogens in localized infections due to the increasing problem of multiantibiotic resistance. Although much has been learned about the mechanisms of microbial killing, there is still uncertainty about whether dyes must bind to and penetrate various classes of microbe in order to produce effective killing after illumination. In this report, we compare the interactions of three antimicrobial photosensitizers: rose bengal (RB), toluidine blue O (TBO), and a poly-L-lysine chlorin(e6) conjugate (pL-ce6) with representative members of three classes of pathogens; Escherichia coli (gram-negative bacteria), Staphylococcus aureus (gram-positive bacteria), Candida albicans (yeast). We compared fluence-dependent cell survival after illumination with the appropriate wavelengths of light before and after extracellular dye had been washed out and used three 10-fold dilutions of cell concentration. pL-ce6 was overall the most powerful photosensitizer, was equally effective with and without washing, and showed a strong dependence on cell concentration. TBO was less effective in all cases after washing, and the dependence on cell concentration was less pronounced. RB was ineffective after washing (except for S. aureus) but still showed a dependence on cell concentration. The overall order of susceptibility was S. aureus>E. coli>C. albicans, but C. albicans cells were 10 to 50 times bigger than the bacteria. We conclude that the number and mass of the cells compete both for available dye binding and for extracellularly generated reactive oxygen species.

Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death.

Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In the second of a series of three reviews, we will discuss the mechanisms that operate in PDT on a cellular level. In Part I [Castano AP, Demidova TN, Hamblin MR. Mechanism in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization. Photodiagn Photodyn Ther 2004;1:279-93] it was shown that one of the most important factors governing the outcome of PDT, is how the photosensitizer (PS) interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. PS can localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes. An explosion of investigation and explorations in the field of cell biology have elucidated many of the pathways that mammalian cells undergo when PS are delivered in tissue culture and subsequently illuminated. There is an acute stress response leading to changes in calcium and lipid metabolism and production of cytokines and stress proteins. Enzymes particularly, protein kinases, are activated and transcription factors are expressed. Many of the cellular responses are centered on mitochondria. These effects frequently lead to induction of apoptosis either by the mitochondrial pathway involving caspases and release of cytochrome c, or by pathways involving ceramide or death receptors. However, under certain circumstances cells subjected to PDT die by necrosis. Although there have been many reports of DNA damage caused by PDT, this is not thought to be an important cell-death pathway. This mechanistic research is expected to lead to optimization of PDT as a tumor treatment, and to rational selection of combination therapies that include PDT as a component.

Mechanisms in photodynamic therapy: Part three-Photosensitizer pharmacokinetics, biodistribution, tumor localization and modes of tumor destruction.

Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as cancer therapy, some of its most successful applications are for non-malignant disease. The majority of mechanistic research into PDT, however, is still directed towards anti-cancer applications. In the final part of series of three reviews, we will cover the possible reasons for the well-known tumor localizing properties of photosensitizers (PS). When PS are injected into the bloodstream they bind to various serum proteins and this can affect their phamacokinetics and biodistribution. Different PS can have very different pharmacokinetics and this can directly affect the illumination parameters. Intravenously injected PS undergo a transition from being bound to serum proteins, then bound to endothelial cells, then bound to the adventitia of the vessels, then bound either to the extracellular matrix or to the cells within the tumor, and finally to being cleared from the tumor by lymphatics or blood vessels, and excreted either by the kidneys or the liver. The effect of PDT on the tumor largely depends at which stage of this continuous process light is delivered. The anti-tumor effects of PDT are divided into three main mechanisms. Powerful anti-vascular effects can lead to thrombosis and hemorrhage in tumor blood vessels that subsequently lead to tumor death via deprivation of oxygen and nutrients. Direct tumor cell death by apoptosis or necrosis can occur if the PS has been allowed to be taken up by tumor cells. Finally the acute inflammation and release of cytokines and stress response proteins induced in the tumor by PDT can lead to an influx of leukocytes that can both contribute to tumor destruction as well as to stimulate the immune system to recognize and destroy tumor cells even at distant locations.

Mechanisms in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization.

The use of non-toxic dyes or photosensitizers (PS) in combination with harmless visible light that is known as photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In a series of three reviews we will discuss the mechanisms that operate in the field of PDT. Part one discusses the recent explosion in discovery and chemical synthesis of new PS. Some guidelines on how to choose an ideal PS for a particular application are presented. The photochemistry and photophysics of PS and the two pathways known as Type I (radicals and reactive oxygen species) and Type II (singlet oxygen) photochemical processes are discussed. To carry out PDT effectively in vivo, it is necessary to ensure sufficient light reaches all the diseased tissue. This involves understanding how light travels within various tissues and the relative effects of absorption and scattering. The fact that most of the PS are also fluorescent allows various optical imaging and monitoring strategies to be combined with PDT. The most important factor governing the outcome of PDT is how the PS interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. Examples of PS that localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes are given. Finally the use of 5-aminolevulinic acid as a natural precursor of the heme biosynthetic pathway, stimulates accumulation of the PS protoporphyrin IX is described.