Antimicrobial Photosensitizers Research Articles

Optimal Photosensitizers for Photodynamic Therapy of Infections Should Kill Bacteria but Spare Neutrophils

Photodynamic therapy (PDT) for localized microbial infections exerts its therapeutic effect both by direct bacterial killing and also by the bactericidal effects of host neutrophils stimulated by PDT. Therefore, PDT-induced damage to neutrophils must be minimized, while direct photoinactivation of bacteria is maintained to maximize the therapeutic efficacy of antimicrobial PDT in vivo. However, there has been no study in which the cytocidal effect of PDT on neutrophils was investigated. In this study, the cytocidal effects of PDT on neutrophils were evaluated using different antimicrobial photosensitizers to find suitable candidate photosensitizers for antimicrobial PDT. PDT on murine peripheral-blood neutrophils was performed in vitro using each photosensitizer at a concentration that exerted a maximum bactericidal effect on methicillin-resistant Staphylococcus aureus, and morphological alteration and viability of neutrophils were studied. Most neutrophils were viable (>80%) after PDT using toluidine blue-O (TB) or methylene blue (MB), while neutrophils showed morphological change and their viabilities were decreased (<70%) after PDT using other photosensitizers (erythrosine B, rose bengal, crystal violet, Photofrin, new methylene blue and Laserphyrin). These results suggest that PDT using TB or MB can preserve host neutrophils while exerting a significant therapeutic effect on in vivo localized microbial infection.

Photodynamic therapy for infections: Clinical applications

Photodynamic therapy (PDT) was discovered over 100 years ago by its ability to kill various microorganisms when the appropriate dye and light were combined in the presence of oxygen. However it is only in relatively recent times that PDT has been studied as a treatment for various types of localized infections. This resurgence of interest has been partly motivated by the alarming increase in drug resistance amongst bacteria and other pathogens. This review will focus on the clinical applications of antimicrobial PDT. The published peer-reviewed literature was reviewed between 1960 and 2011. The basics of antimicrobial PDT are discussed. Clinical applications of antimicrobial PDT to localized viral infections caused by herpes and papilloma viruses, and nonviral dermatological infections such as acne and other yeast, fungal and bacterial skin infections are covered. PDT has been used to treat bacterial infections in brain abscesses and non-healing ulcers. PDT for dental infections including periodontitis and endodontics has been well studied. PDT has also been used for cutaneous Leishmaniasis. Clinical trials of PDT and blue light alone therapy for gastric Helicobacter pylori infection are also covered. As yet clinical PDT for infections has been mainly in the field of dermatology using 5-aminolevulanic acid and in dentistry using phenothiazinium dyes. We expect more to see applications of PDT to more challenging infections using advanced antimicrobial photosensitizers targeted to microbial cells in the years to come.

Substituted zinc phthalocyanine as an antimicrobial photosensitizer for periodontitis treatment

In the last decades the worldwide rise in antibiotic resistance has intensified the development of new antimicrobial agents. Photodynamic antimicrobial chemotherapy (PACT) has been used successfully to inactivate bacteria. We herein report a new zinc phthalocyanine based photosensitizer conjugated with polylysine moiety ( ZnPc-PL ). This photosensitizer significantly inactivated Porphyromonas gingivalis, the primary pathogenic bacteria responsible for periodontitis. No obvious phototoxicity was found to either mammalian bone marrow stromal cells (BMSC) or human periodontal ligament cells (HPDLC), indicating the high selectivity of ZnPc -PL toward bacteria. Furthermore, we established an experimental periodontitis model on beagle dogs to test the antimicrobial efficacy in vivo. The amount of gingival crevicular fluid (GCF) and the activity of crevicular fluid aspartate aminotransferase (AST) were monitored and were found to reduce significantly in the ZnPc-PL treated group compared to the controls (laser only and no treatment). In addition, PACT with ZnPc -PL caused a reduction in the bacterial burden by 100-fold compared to controls. Taken together, these findings suggest ZnPc-PL is a promising antimicrobial photosensitizer for the treatment of periodontal diseases.

Photodynamic therapy with a cationic functionalized fullerene rescues mice from fatal wound infections.

Fullerenes are under intensive study for potential biomedical applications. We have previously reported that a C60 fullerene functionalized with three dimethylpyrrolidinium groups (BF6) is a highly active broad-spectrum antimicrobial photosensitizer in vitro when combined with white-light illumination. We asked whether this high degree of in vitro activity would translate into an in vivo therapeutic effect in two potentially lethal mouse models of infected wounds. We used stable bioluminescent bacteria and a low light imaging system to follow the progress of the infection noninvasively in real time. An excisional wound on the mouse back was contaminated with one of two bioluminescent Gram-negative species, Proteus mirabilis (2.5 × 10(7) cells) and Pseudomonas aeruginosa (5 × 10(6) cells). A solution of BF6 was placed into the wound followed by delivery of up to 180 J/cm(2) of broadband white light (400-700 nm). In both cases there was a light-dose-dependent reduction of bioluminescence from the wound not observed in control groups (light alone or BF6 alone). Fullerene-mediated photodynamic therapy of mice infected with P. mirabilis led to 82% survival compared with 8% survival without treatment (p < 0.001). Photodynamic therapy of mice infected with highly virulent P. aeruginosa did not lead to survival, but when photodynamic therapy was combined with a suboptimal dose of the antibiotic tobramycin (6 mg/kg for 1 day) there was a synergistic therapeutic effect with a survival of 60% compared with a survival of 20% with tobramycin alone (p < 0.01). These data suggest that cationic fullerenes have clinical potential as an antimicrobial photosensitizer for superficial infections where red light is not needed to penetrate tissue.

Stable synthetic cationic bacteriochlorins as selective antimicrobial photosensitizers.

Photodynamic inactivation is a rapidly developing antimicrobial treatment that employs a nontoxic photoactivatable dye or photosensitizer in combination with harmless visible light to generate reactive oxygen species that are toxic to cells. Tetrapyrroles (e.g., porphyrins, chlorins, bacteriochlorins) are a class of photosensitizers that exhibit promising characteristics to serve as broad-spectrum antimicrobials. In order to bind to and efficiently penetrate into all classes of microbial cells, tetrapyrroles should have structures that contain (i) one or more cationic charge(s) or (ii) a basic group. In this report, we investigate the use of new stable synthetic bacteriochlorins that have a strong absorption band in the range 720 to 740 nm, which is in the near-infrared spectral region. Four bacteriochlorins with 2, 4, or 6 quaternized ammonium groups or 2 basic amine groups were compared for light-mediated killing against a gram-positive bacterium (Staphylococcus aureus), a gram-negative bacterium (Escherichia coli), and a dimorphic fungal yeast (Candida albicans). Selectivity was assessed by determining phototoxicity against human HeLa cancer cells under the same conditions. All four compounds were highly active (6 logs of killing at 1 microM or less) against S. aureus and showed selectivity for bacteria over human cells. Increasing the cationic charge increased activity against E. coli. Only the compound with basic groups was highly active against C. albicans. Supporting photochemical and theoretical characterization studies indicate that (i) the four bacteriochlorins have comparable photophysical features in homogeneous solution and (ii) the anticipated redox characteristics do not correlate with cell-killing ability. These results support the interpretation that the disparate biological activities observed stem from cellular binding and localization effects rather than intrinsic electronic properties. These findings further establish cationic bacteriochlorins as extremely active and selective near-infrared activated antimicrobial photosensitizers, and the results provide fundamental information on structure-activity relationships for antimicrobial photosensitizers.

Innovative cationic fullerenes as broad-spectrum light-activated antimicrobials

Photodynamic inactivation is a rapidly developing antimicrobial technology that combines a nontoxic photoactivatable dye or photosensitizer in combination with harmless visible light of the correct wavelength to excite the dye to its reactive-triplet state that will then generate reactive oxygen species that are highly toxic to cells. Buckminsterfullerenes are closed-cage molecules entirely composed of sp2-hybridized carbon atoms, and although their main absorption is in the UV, they also absorb visible light and have a long-lived triplet state. When C 60 fullerene is derivatized with cationic functional groups it forms molecules that are more water-soluble and can mediate photodynamic therapy efficiently upon illumination; moreover, cationic fullerenes can selectively bind to microbial cells. In this report we describe the synthesis and characterization of several new cationic fullerenes. Their relative effectiveness as broad-spectrum antimicrobial photosensitizers against gram-positive and gram-negative bacteria, and a fungal yeast was determined by quantitative structure-function relationships. From the Clinical Editor Photodynamic inactivation (PDI) is a rapidly developing antimicrobial technology in which a non-toxic photoactivatable dye or photosensitizer is excited with harmless visible light to its reactive state, where it will generate highly toxic reactive oxygen species. Buckminsterfullerenes derivatized with cationic functional groups form molecules that are water-soluble and mediate PDI efficiently. These fullerenes can also selectively bind to microbial cells. Several new cationic fullerenes are presented in this paper, and their efficacy against Gram-positive, Gram-negative bacteria, and a fungal yeast is also demonstrated.

Synthesis and Properties of Benzo[a]phenoxazinium Chalcogen Analogues as Novel Broad-Spectrum Antimicrobial Photosensitizers.

Synthesis and Properties of Benzo[<i>a</i>]phenoxazinium Chalcogen Analogues as Novel Broad-Spectrum Antimicrobial Photosensitizers.

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.

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.

A novel synthesis of 5,15-trimethylammonium substituted porphyrins and their evaluation as potential antimicrobial photosensitizers

The MacDonald's [2 + 2] condensation of unsubstituted dipyrromethane and 3- or 4-hydroxyalkoxybenzoaldehydes afforded 5,15-meso-hydroxyalkoxyphenylporphyrins. The hydroxyl groups on the side chains were then transformed to tertiary amines via synthesis of the mesylate and substitution with the appropriate amines or aminoalcohols. Methylation of such products afforded a series of cationic porphyrins which have been tested in vitro as photodynamic agents against microorganisms, showing high antimicrobial activity.

Antimicrobial DNA-binding Photosensitizers from the Common Rush, Juncus effusus¶

Our continuing survey of phototoxins from higher plants has led to the isolation and identification from the common rush, Juncus effusus L., of the phenanthrene, dehydroeffusol (1), and the dihydrophenanthrene, juncusol (2), compounds that display enhanced antimicrobial activities in light. The antimicrobial activities (minimum inhibitory concentrations) for these compounds against methicillin-resistant and -sensitive Staphylococcus aureus and Candida albicans were increased 16- and two-fold, respectively, by irradiation with ultraviolet A (UVA). Photosensitized DNA-binding activities (as possible covalent bond formation) of these compounds were determined by using restriction enzymes and a specially prepared 1.5 kb DNA fragment. Under UVA irradiation, dehydroeffusol strongly inhibited all the restriction enzymes (KpnI, XbaI, PmeI, DraI, PacI and BciVI) that have at least one 5′-TpA sequence in their recognition sites. Weak inhibitions were found for the restriction enzymes EcoRI, SacI, BamHI, SalI, PstI and HindIII, which do not possess a 5′-TpA sequence at their restriction sites and the restriction site sequences of which consist of all bases, A, T, G and C. Trace or no inhibition was found for AscI and SmaI, the restriction site sequences of which are composed of only C and G. The results indicate the necessity of thymine (adenine) for the photosensitized DNA-binding activity of dehydroeffusol. A strong inhibition against SphI, which does not have a 5′-TpA sequence in the restriction sequence, indicates that there are possibly other binding sequence(s) for dehydroeffusol. With juncusol and UVA, strong inhibitions for KpnI and BciVI and trace inhibitions for PacI, XbaI, PmeI and DraI were found. This result also showed a preference of juncusol for 5′-TpA, but the preference could be more selective than that of dehydroeffusol depending on the surrounding sequences of 5′-TpA in the respective restriction sites. A strong inhibition of SphI by juncusol with UVA also indicated the existence of an unknown binding sequence for this compound. Generally, the DNA-binding activity of this compound was weaker than that of dehydroeffusol.

Antimicrobial DNA-binding photosensitizers from the common rush, Juncus effusus.

Our continuing survey of phototoxins from higher plants has led to the isolation and identification from the common rush, Juncus effusus L., of the phenanthrene, dehydroeffusol (1), and the dihydrophenanthrene, juncusol (2), compounds that display enhanced antimicrobial activities in light. The antimicrobial activities (minimum inhibitory concentrations) for these compounds against methicillin-resistant and -sensitive Staphylococcus aureus and Candida albicans were increased 16- and two-fold, respectively, by irradiation with ultraviolet A (UVA). Photosensitized DNA-binding activities (as possible covalent bond formation) of these compounds were determined by using restriction enzymes and a specially prepared 1.5 kb DNA fragment. Under UVA irradiation, dehydroeffusol strongly inhibited all the restriction enzymes (KpnI, XbaI, PmeI, DraI, PacI and BciVI) that have at least one 5'-TpA sequence in their recognition sites. Weak inhibitions were found for the restriction enzymes EcoRI, SacI, BamHI, SalI, PstI and HindIII, which do not possess a 5'-TpA sequence at their restriction sites and the restriction site sequences of which consist of all bases, A, T, G and C. Trace or no inhibition was found for AscI and SmaI, the restriction site sequences of which are composed of only C and G. The results indicate the necessity of thymine (adenine) for the photosensitized DNA-binding activity of dehydroeffusol. A strong inhibition against SphI, which does not have a 5'-TpA sequence in the restriction sequence, indicates that there are possibly other binding sequence(s) for dehydroeffusol. With juncusol and UVA, strong inhibitions for KpnI and BciVI and trace inhibitions for PacI, XbaI, PmeI and DraI were found. This result also showed a preference of juncusol for 5'-TpA, but the preference could be more selective than that of dehydroeffusol depending on the surrounding sequences of 5'-TpA in the respective restriction sites. A strong inhibition of SphI by juncusol with UVA also indicated the existence of an unknown binding sequence for this compound. Generally, the DNA-binding activity of this compound was weaker than that of dehydroeffusol.