Polylysine–porphycene conjugates as efficient photosensitizers for the inactivation of microbial pathogens

211

138

The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to the killing of gram-positive antibiotic-resistant bacteria of the skin uses light in combination with a photosensitizer to induce a phototoxic reaction. Different concentrations (0 to 100 M) of porphyrin-based photosensitizers (CTP1, XF70, and XF73) and different incubation times (5 min, 1 h, and 4 h) were used to determine phototoxicity against two methicillin-resistant Staphylococcus aureus strains, one methicillin-sensitive S. aureus strain, one methicillin-resistant Staphylococcus epidermidis strain, one Escherichia coli strain, and human keratinocytes and fibroblasts. Incubation with 0.005 M XF70 or XF73, followed by illumination, yielded a 3-log 10 (>99.9%) decrease in the viable cell numbers of all staphylococcal strains, indicating that the XF drugs have high degrees of potency against gram-positive bacteria and also that the activities of these novel drugs are independent of the antibiotic resistance pattern of the staphylococci examined. CTP1 was less potent against the staphylococci under the same conditions. At 0.005 M, XF70 and XF73 demonstrated no toxicity toward fibroblasts or keratinocytes. No inactivation of E. coli was detected at this concentration. XF73 was confirmed to act via a reactive oxygen species from the results of studies with sodium azide (a quencher of singlet oxygen), which reduced the killing of both eukaryotic and prokaryotic cells. When a quencher of superoxide anion and the hydroxyl radical was used, cell killing was not inhibited. These results demonstrate that the porphyrin-based photosensitizers had concentration-dependent differences in their efficacies of killing of methicillin-resistant staphylococcal strains via reactive oxygen species without harming eukaryotic cells at the same concentrations.

Section: Discussionsupporting

confidence: 91%

Photodynamic Effects of Novel XF Porphyrin Derivatives on Prokaryotic and Eukaryotic Cells

Maisch¹,

Bosl²,

Szeimies³

et al. 2005

211

138

“…Now we have shown that C. albicans is the hardest to kill with all three PS and that TBO comes closest to having equivalent efficacy for Candida as it has for bacteria. In agreement with a report by Polo et al (25), we have shown that a polycationic PS conjugate with pL is also highly effective against fungal cells as well as bacteria.…”

Section: Vol 49 2005 Microbial Photoinactivation and Cell-dye Affinsupporting

confidence: 93%

Effect of Cell-Photosensitizer Binding and Cell Density on Microbial Photoinactivation

Demidova

Hamblin

2005

387

285

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.

“…Polo and coworkers used conjugates between pL and porphycenes with significant phototoxic activity against gram-negative bacteria (40). In a subsequent report (25) these authors showed that several strains responsible for periodontal disease were efficiently inactivated by visible-light irradiation in the presence of porphycene-pL conjugates.…”

mentioning

confidence: 99%

Protease-Stable Polycationic Photosensitizer Conjugates between Polyethyleneimine and Chlorin(e6) for Broad-Spectrum Antimicrobial Photoinactivation

Tegos

Anbe

Yang

et al. 2006

161

146

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 gramnegative 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 therapy (PDT) employs a nontoxic dye termed a photosensitizer (PS) and low-intensity visible light, which in the presence of oxygen produce cytotoxic species (4-6). PDT has the advantage of dual selectivity in that the PS can be targeted to its destination cell or tissue, and in addition the illumination can be spatially directed to the lesion. PDT was originally discovered over 100 years ago by its effect on microorganisms (35) but since then has been principally developed as a treatment for cancer (11) and age-related macular degeneration (3).Because of the well-known increase in multiantibiotic resistance among pathogenic microbes of all classes, PDT has attracted attention as a possible treatment for localized infections (9, 14, 46). It is known that gram-negative bacteria are resistant to PDT with many commonly used PS that will readily lead to phototoxicity for gram-positive species (29) and that PS bearing a cationic charge (33) or the use of agents that increase the permeability of the outer membrane will increase the efficacy of killing of gram-negative organisms (29).In 1997 our laboratory formed the hypothesis that by covalently conjugating a suitable PS such as chlorin(e6) (ce6) to a poly-L-lysine (pL) chain, a bacterially targeted PS delivery vehicle could be constructed that would efficiently inactivate both gram-positive and gram-negative species (42). Because the resulting polycationic entity is a macromolecule, it is taken up by mammalian cells by th...