STRUCTURE-ACTIVITY RELATIONSHIP OF PORPHINES FOR PHOTOINACTIVATION OF BACTERIA

Abstract— The antibacterial photodynamic effects of uncharged ( o -tetrahydroxyphenyl porphine [THPP], m -THPP and p -THPP), cationic (5,10,15,20-tetra[4- N -methylpyridyllporphine [TMPyP]) and anionic (5,10,15,20-tetra[4-sulfonatophenyl porphine] [TPPS₄]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane-disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram-positive bacteria but only marginally effective on gram-negative cells, whereas TPPS₄ showed no activity on either gram-positive or gram-negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram-positive target cells. For TPPS₄, the inactivity toward gram-positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram-negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure-activity relationships of these porphines are affected by their net charge and by aggregation.     

DNA Damage and Apoptosis Induced by Photosensitization of 5,10,15,20-Tetrakis (N-methyl-4-pyridyl)-21H,23H-porphyrin via Singlet Oxygen Generation

Cancer photodynamic therapy (PDT) requires photosensitizers that efficiently and selectively destroy tumor cells. We investigated 5,10,15,20-tetrakis ( N -methyl-4-pyridyl)-21 H ,23 H -porphyrin (TMPyP) as a potential cancer treatment. Confocal fluorescence microscopy showed that TMPyP was localized in the nuclei, whereas 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PPIX) was localized diffusely in the cytoplasm of human leukemia (HL-60) cells. In HL-60 cells under UVA irradiation, TMPyP effectively induced apoptosis. Moreover, 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative product of 2'-deoxyguanosine, was accumulated in the DNA of cells treated with photoirradiated TMPyP, whereas only small amounts were observed in ALA-treated cells in the presence of UVA light. TMPyP and UVA caused extensive damage at every guanine residue in DNA fragments obtained from the human p 53 tumor suppressor gene and the c-Ha- ras -1 proto-oncogene, whereas PPIX induced little DNA damage under these conditions. Electron spin resonance spectroscopy using a singlet oxygen (¹O₂) probe and D₂O showed that photoexcited TMPyP generated ¹O₂. These results suggest that photoexcited TMPyP reacts with oxygen to generate ¹O₂, which in turn, oxidizes guanine residues. Taken together, the results demonstrated that TMPyP was localized in the nucleus where it was photosensitized to induce DNA damage, suggesting that TMPyP may have clinical utility as a nucleus-targeted PDT.     

Fabrication and characterization of a α,β,γ,δ-Tetrakis(1-methylpyridinium-4-yl)porphine/silica nanocomposite thin-layer membrane for detection of ppb-level heavy metal ions

A new detection membrane for filtration enrichment combined with colorimetric determination of Cd(II), Zn(II), Pb(II) and Cu(II) ions is presented. We have demonstrated the use of a dye nanoparticle coated test strip (DNTS) structured with a reagent layer for on-site analysis of trace metal ions. In this study, a [TMPyP/SA] DNTS coated with a nanocomposite layer (average thickness: 5.39 μm) of α,β,γ,δ-Tetrakis(1-methylpyridinium-4-yl)porphine (TMPyP) and silica-SA on the top surface of a cellulose ester membrane filter was fabricated by a simple filtration of an aqueous TMPyP/silica-SA nanocomposite dispersion through a membrane filter. The nanocomposite formation of cationic TMPyP and negatively charged colloidal SA (9–80 nm) was based on electrostatic interaction and was confirmed in the 120–800 nm diameter range by a dynamic light scattering photometer (DLS). To optimize the DNTS nanocomposite layer, surface uniformity, mechanical strength, the percent retention of TMPyP, and sensitivity to Cd(II) detection for six DNTSs with five different types of silica were examined. A half[TMPyP/SA] DNTS with an average layer thickness of 2.60 μm, which was prepared by controlling the amount of TMPyP and SA, demonstrated the highest sensitivity to Cd(II) ion because it had the lowest background absorbance. In addition, factors that affected the percent retention of TMPyP, such as pH and TMPyP/SA ratio, were determined. More than 99% of the TMPyP was retained on a membrane filter at pH 7.8 with a TMPyP and SA concentration of 2 × 10⁻⁵ M and 4 × 10⁻⁵ wt%, respectively. Filtration enrichment of 100 mL of an aqueous solution containing Cd(II), Zn(II), and Pb(II) at ppb levels was achieved by concentrating the metal ions in a nanocomposite layer (the effective TMPyP area was 1.77 cm², pH 10.2). The signaling surface changed from a brown color to green when the ions were captured. The percent extraction for metal ions on a half[TMPyP/SA] DNTS were estimated by TLC scanning and ICP-MS. It was observed that, when using the half[TMPyP/SA] DNTS, Cd(II) concentrations as low as 1 ppb were detectable at a filtration rate of 4.0–5.0 mL min⁻¹.     

Photodamage in a Mitochondrial Membrane Model Modulated by the Topology of Cationic and Anionic Meso‐Tetrakis Porphyrin Free Bases

The photodynamic effects of the cationic TMPyP (meso‐tetrakis [N‐methyl‐4‐pyridyl]porphyrin) and the anionic TPPS4 (meso‐tetrakis[4‐sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the influence of phorphyrin binding on lipid damage. Electronic absorption spectroscopy and zeta potential measurements demonstrated that only TMPyP binds to PC/CL large unilamellar vesicles (LUVs). The photodamage after irradiation with visible light was analyzed by dosages of lipid peroxides (LOOH) and thiobarbituric reactive substance and by a contrast phase image of the giant unilamellar vesicles (GUVs). Damage to LUVs and GUVs promoted by TMPyP and TPPS4 were qualitatively and quantitatively different. The cationic porphyrin promoted damage more extensive and faster. The increase in LOOH was higher in the presence of D₂O, and was impaired by sodium azide and sorbic acid. The effect of D₂O was higher for TPPS4 as the photosensitizer. The use of DCFH demonstrated that liposomes prevent the photobleaching of TMPyP. The results are consistent with a more stable TMPyP that generates long‐lived singlet oxygen preferentially partitioned in the bilayer. Conversely, TPPS4 generates singlet oxygen in the bulk whose lifetime is increased in D₂O. Therefore, the affinity of the porphyrin to the membrane modulates the rate, type and degree of lipid damage.     

Effect of High Intensity Blue Light on Fusobacterium nucleatum Membrane Integrity

Oral malodour is considered to be caused mainly by the production of volatile sulfide compounds (VSC) by anaerobic gram-negative oral bacteria. Previous studies showed that these bacteria were susceptible to blue light phototoxicity mediated by the production of reactive oxygen species (ROS). In the present study, we tested the effect of blue light on the integrity Fusobacterium nucleatum's membrane, cellular proteins and DNA. Bacterial samples were exposed to high intensity blue light for 0, 70, 140 and 280 s (i.e. fluences of 0, 96, 192 and 384 J cm⁻², respectively). Following light exposure, bacterial samples were examined for membrane damage using fluorescence microscopy, intra-cellular protein analysis using electrophoresis (SDS-PAGE) and DNA fragmentation using ultra–filtration. Results showed that the increasing exposure of bacterial samples to blue light caused increased membrane permeability concomitant with a reduction in intra-cellular proteins and DNA fragments content. These results suggest that membrane damage is the main effect of high intensity blue light exposure on malodour producing bacteria.     

Enhanced Bactericidal Activity of Modified Titania in Sunlight against Pseudomonas aeruginosa,a Water-Borne Pathogen

Photocatalyst-mediated inactivations generate reactive oxygen species and OH radicals, which induce oxidative destruction of membrane integrity, causing damage to membrane phospholipids of gram negative bacteria like Pseudomonas aeruginosa. Nanosized TiO₂ was synthesized by gel to crystalline conversion and Zr-doped TiO₂ was synthesized by pulverization using appropriate precursor. The doped nanocrystals retained the anatase phase with a marginal increase in crystallite size, averaging at 25 nm. SEM–EDX analysis of the doped sample depicts the substantial growth of grain size with 1.33 atomic weight % of zirconium. The created electron states in the doped sample act as charge carrier traps suppressing recombination which later detraps the same to the surface of the catalyst causing enhanced interfacial charge transfer. Zr-doped TiO₂ at the molecular scale exhibits better photocatalytic activity with lower bandgap energy that can respond to visible light. The redshift caused by the dopants in absorption spectra of TiO₂ facilitated the nonintrinsic sample to exhibit nearly 2-fold enhancement of photoinactivation in sunlight. Extent of photoinactivation of P. aeruginosa was observed to be complete (100%) within 150 min of sunlight exposure in the presence of modified TiO₂.     

In vivo effects of porphyrins on bacterial DNA.

The DNA damage in intact Staphylococcus aureus and E. coli cells induced by photosensitized deuteroporphyrin or hemin is described. Treatment of S. aureus cultures with hemin or photosensitized deuteroporphyrin (Dp) caused time-dependent changes in the plasmidial DNA profiles. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected by hemin and by photosensitized Dp, since its degradation products were detected after exposing the bacterial cells to the porphyrin drugs. Photosensitization of E. coli cells, pretreated with Dp and polymyxin B nonapeptide (PMBNP), also resulted in plasmidial damage. No such damage occurred when E. coli cultures were treated with hemin and PMBNP. The above results can be tightly correlated with the antimicrobial action of porphyrins. Their damage to the bacterial DNA seems to reflect one of the in vivo effects of these porphyrins.     

FLUORESCENCE SPECTRAL CHANGES OF HEMATOPORPHYRIN DERIVATIVE UPON BINDING TO LIPID VESICLES, Staphylococcus aureus AND Escherichia coli CELLS

Abstract— The binding of hematoporphyrin derivated (Hpd) to lipid vesicles and bacterial membranes was determined by fluorescence spectroscopy. The fluorescence measurements of Hpd in aqueous solutions showed two bands at 613 and 677 nm. In lipid environments of lecithin vesicles the fluorescence spectrum was shifted to 631 and 692 nm, respectively. Hpd was rapidly bound to the cell membrane of Staphylococcus aureus while much less binding occurred in the presence of Escherichia coli. At the same time, spheroplasts of both bacteria were shown to bind Hpd to a similar extent. These results are well correlated with the photoinactivation of the gram positive bacteria with Hpd while the gram negative cells were shown to be resistant. The pH dependence of both Hpd binding to S. aureus as well as the photodynamic inhibitory effect of the same bacteria are similar. It is concluded that the segregation of Hpd to the cell membrane is a prerequisite for its photodynamic effect.     

Xanthene dyes induce membrane permeabilization of bacteria and erythrocytes by photoinactivation

We analyzed the photoinactivation of the membrane functions of bacteria and erythrocytes induced by xanthene dyes. The dyes tested were rose bengal, phloxine B, erythrosine B and eosin B. These dyes induced the leakage of K(+) from Staphylococcus aureus cells within minutes of photoirradiation, in the order of rose bengal > phloxine B > erythrosine B > eosin B. The ability of dyes to inhibit respiration was weak, except for rose bengal, and the dyes dissipated the membrane potential in similar time traces with changes in K(+) permeability. The xanthene dyes also induced the leakage of K(+) from bovine erythrocytes upon photoirradiation in the same order as that observed with bacteria. Furthermore, we found that the ability to cause the leakage of K(+) from erythrocytes was associated with dye-induced morphological changes, forming a crenated form from the normal discoid. These results are discussed in connection with the ability of xanthene dyes to generate singlet oxygen and bind to bacterial cells, and further compared with the actions of cationic porphyrins, which induced photoinactivation of bacteria through respiratory inhibition.     

Cytochrome b6/f complex as an indigenous photodynamic generator of singlet oxygen in thylakoid membranes

Possible association of photodynamic sensitization by cytochrome b6/f complex (cyt b6/f) via singlet oxygen (1O2) mechanism with photoinhibition damage to photosystem II (PS II) was studied using such subthylakoid preparations as photosystem I (PS I) particles, PS II core complex and cyt b6/f from spinach leaves. Upon exposure to bright light, PS II core complex lost photosynthetic electron transport activity to a certain extent, whose-spectral dependence implied that pheophytin a is likely involved in photoinactivation of PS II core complex in itself. The presence of PS I particles exerted virtually no effect on PS II core photoinactivation. However, the inclusion of cyt b6/f in samples resulted in a marked exacerbation of the photoinactivation, particularly in UV-A and blue light. Such effect of cyt b6/f was suppressed by azide and enhanced by the medium deuteration. Photogeneration of 1O2 from cyt b6/f was confirmed by ESR and spectrophotometry, chemically trapping 1O2. Action spectra for both 1O2 photoproduction and PS II core photoinactivation by cyt b6/f bore a close resemblance to each other, seemingly carrying the absorption characteristics of the Rieske Fe-S protein. A complex deficient in the Rieske protein prepared from intact cyt b6/f showed virtually no generation of 1O2 in light, whereas an efficient photoformation of 1O2 was seen in the Rieske protein preparation. The results suggest that cyt b6/f, rather specifically the Rieske center, may play a prominent role in photoinhibition processes through type II photosensitization in thylakoids.     

Lethal Effect of Photodynamic Treatment on Persister Bacteria

Persister bacteria tolerate bactericidal antibiotics due to transient and reversible phenotypic changes. As these bacteria can limit the effectiveness of antibiotics to eradicate certain infections, their elimination is a relevant issue. Photodynamic therapy seems suitable for this purpose, but phenotypic tolerance to it has also been reported for Pseudomonas aeruginosa . To test whether any phenotypic feature could confer tolerance against both antibiotics and photoinactivation, survivors from exposures to light in the presence of methylene blue were treated with ofloxacin, an antibiotic effective on nongrowing bacteria. Susceptibility to ofloxacin was normal in these bacteria in spite of their increased ability to survive photodynamic inactivation, suggesting the absence of cross‐tolerance. It thus seemed possible to use one of these treatments to eliminate bacteria which had phenotypic tolerance to the other. To test this strategy, persister bacteria emerging from ofloxacin treatments were submitted to the action of light and methylene blue while the antibiotic remained in the bacterial suspension. Persisters lost their clonogenic ability under these conditions and the effects of the treatments seemed to be synergistic. These observations suggest that photodynamic antimicrobial therapy could be used as a complement to antibiotic treatments to eliminate persister bacteria from localized infections.     

In situ SUSCEPTIBILITIES OF PHOTOSYSTEMS I AND II TO PHOTOSENSITIZED DEACTIVATION via SINGLET OXYGEN MECHANISM

Abstract— A comparative study was carried out on the in situ susceptibilities to photoinactivation of the photosystem I (PS I) and II (PS II) complexes of spinach thylakoids treated with efficient type II sensitizers. While the presence of the exogenous sensitizers caused a substantial increase in the extent of photoinactivation of whole chain electron transport, it did not affect PS I activity of thylakoids in light but exerted an enhanced photoinactivating effect only on PS II. The measurements of the action spectrum for the inhibition of PS II activity of the sensitizer-incorporated thylakoids and that for the generation of singlet oxygen (¹O₂) from them revealed that photosensitized inactivation of PS II is directly related to the photoproduction of ¹O₂ in thylakoid membranes. The results obtained in the present work clearly demonstrate an exceptional sensitivity of PS II to ¹O₂, providing circumstantial evidence that high light-induced damage to PS II may result from photosensitization reactions mediated by ¹O₂, which is not necessarily produced within the PS II complex.     

Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.     

Photodynamic effects of antioxidant substituted porphyrin photosensitizers on gram-positive and -negative bacterial

Photodynamic treatment of the gram-negative bacteria Escherichia coli B and Acinetobacter baumannii and the gram-positive bacterium Staphylococcus aureus was performed using two newly devised and synthesized antioxidant carrier photosensitizers (antioxidant carrier sensitizers-2 [ACS-2] and antioxidant carrier sensitizers-3 [ACS-3]), which are butyl hydroxy toluene and propyl gallate substituted haematoporphyrins, respectively. It was found that ACS-2 is less reactive than other photosensitizers previously used for the same purpose, whereas ACS-3 is very effective against the multidrug-resistant bacterium A. baumannii, causing its complete eradication at a low fluence (approximately 7.5 J/cm2) of blue light (407-420 nm) and a low concentration (10 microM). At a higher fluence (approximately 37.5 J/cm2) complete eradication of E. coli B can be obtained under the same conditions. Furthermore, X-ray microanalysis and ultrastructural changes indicate that ACS-3, especially in the case of photodynamic treatment of A. baumannii, interferes with membrane functions and causes the inactivation of the bacterium. ACS-3 may be suggested as a specific photosensitization agent for photoinactivation of gram-negative bacteria.     

CYTOPROTECTION AGAINST MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF THE Na+,K+-ADENOSINE TRIPHOSPHATASE IN LEUKEMIA CELLS: GLUTATHIONE AND SELENOPEROXIDASE INVOLVEMENT

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied controls (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na⁺,K⁺-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na⁺, K⁺-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photoinactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na⁺,K⁺-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na⁺,K⁺-ATPase, but not AChE. This is consistent with the fact that Na⁺, K⁺-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.     

Kinetics and efficiency displayed by supported and suspended TiO2 catalysts applied to the disinfection of Escherichia coli

TiO₂-mediated photocatalysis is widely used in a variety of applications and products in the environmental and energy fields, including photoelectrochemical conversion, self-cleaning surfaces, and especially water purification systems. The dimensionality of the structure of a TiO₂ material can affect its properties, functions, and more specifically, its photocatalytic performance. In this work, the photocatalytic inactivation of Gram-negative Escherichia coli using three photocatalysts, differing in their structure and other characteristics, was studied in a batch reactor under UVA light. The aim was to establish the disinfection efficiency of solid TiO₂ compared with that of suspended catalysts, widely considered as reference cases for photocatalytic water disinfection. The bacterial inactivation profiles obtained showed that: (1) the photoinactivation was exclusively related to the quantity of photons retained per unit of treated volume, irrespective of the characteristics of the photocatalyst and the emitted light flux densities; (2) across the whole UV light range studied, each of the photocatalytic solids was able to achieve more than 2 log bacterial inactivation with less than 2 h UV irradiation; (3) none of the used catalysts achieved a total bacterial disinfection during the treatment time. For each of the catalysts the quantum yield has been assessed in terms of disinfection efficiency, the 2D material showed almost the same performance as those of suspended catalysts. This catalyst is promising for supported photocatalysis applications.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

Effect of Toxic Metal Ions on Photosensitized Singlet Oxygen Generation for Photodegradation of Polyaromatic Hydrocarbon Derivatives and Inactivation of Escherichia coli

Here, we report an experimental study of the effect of toxic metal ions on photosensitized singlet oxygen generation for photodegradation of PAH derivatives, Anthracene‐9,10‐dipropionic acid disodium salt (ADPA) and 1,5‐dihydroxynapthalene (DHN) and photoinactivation of Escherichia coli bacteria by using cationic meso‐tetra(N‐methyl‐4‐pyridyl)porphine tetrachloride (TMPyP) as a singlet oxygen photosensitizer. Three s‐block metals ions, such as Na⁺, K⁺ and Ca²⁺ and five toxic metals such as Cd²⁺, Cu²⁺, Hg²⁺, Zn²⁺ and Pb²⁺ were studied. The s‐block metal ions showed no change in the rate of photodegradation of ADPA or DHN by TMPyP, whereas a dramatic change in the photodegradation of ADPA and DHN was observed in the presence of toxic metals. The maximum photodegradation rate constants of ADPA and DHN were observed for Cd²⁺ ions [(3.91 ± 0.20) × 10^?3 s^?1 and (7.18 ± 0.35) × 10^?4 s^?1, respectively]. Strikingly, the photodegradation of ADPA and DHN was almost completely inhibited in the presence of Hg²⁺ ions and Cu²⁺ ions. A complete inhibition of growth of E. coli was observed upon visible light irradiation of E. coli solutions with TMPyP and toxic metal ions particularly, Cd²⁺, Hg²⁺, Zn²⁺ and Pb²⁺ ions, except for Cu²⁺ ions where a significantly slow inhibition of E. coli's growth was observed.     

Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. In this study we report on the first use of a photochemical approach to destroy bacteria infecting a wound in an animal model. Following topical application, a targeted polycationic photosensitizer conjugate between poly-L-lysine and chlorin(e6) penetrated the gram (-) outer bacterial membrane, and subsequent activation with 660 nm laser light rapidly killed Escherichia coli infecting excisional wounds in mice. To facilitate real-time monitoring of infection, we used bacteria that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low-light imaging system. There was a light-dose dependent loss of luminescence in the wound treated with conjugate and light, not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the photodynamic treatment did not damage the host tissue. Our study points to the possible use of this methodology in the rapid control of wounds and other localized infections.