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The antibacterial photodynamic effects of uncharged (o-tetrahydroxyphenyl porphine [THPP], m-THPP and p-THPP), cationic (5,10,15,20-tetra[4-N-methylpyridyl]porphine [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.

1. 1. The immediate and fast ionic fluxes in Friend erythroleukemic cells (FELC), erythrocytes and Staphylococcus aureus during short intervals of porphyrin mediated photosensitization were determined uniquely by X-ray microanalysis (XRMA) combined with electron microscopy. 2. 2. Photodynamic inactivation of FELC was mediated by either endogenous protoporphyrin induced by 5-amino levulinic acid (5-ALA), or Photofrin-II. We describe the predominant phenomena of >85% K-loss within 2–10 min of photoactivation. However the accompanied Na inflow and the collapse of the cellular balance of elemental-composition were inconsistent and acted as a function of cell damage. 3. 3. Erythrocytes treated with hematoporphyrin (HP) lost most of their intracellular K yet instantly gained Na. Nevertheless the K/Na molar ratio of the control erythrocytes was nearly 12/1 while after photosensitization and K loss it changed to 1/1. 4. 4. The S. aureus bacteria photosensitized with HP showed entire K-loss as well as marked Na efflux which increased with irradiation time; this was accompanied by the decline of other cell elements. 5. 5. The prevailing K loss in FELC, erythrocytes and bacteria during the first minutes of photosensitization is deduced to be an immediate primary consequence of the photodynamic effect, while other ionic changes are joined in order with the development of cellular damage.

The combined antibacterial activity of various porphyrins with hemin on Gram-positive bacteria was studied. Protoporphyrin, hematoporphyrin derivative and deuteroporphyrin show only a marginal inhibitory effect in the dark. However, hemin has a strong cytotoxic effect which is independent of illumination and is equally strong in the dark. The disadvantage of hemin treatment is that it is temporary. In this study, we have demonstrated that a combination of deuteroporphyrin and hemin has a unique cytotoxic activity on Staphylococcus aureus, Streptococcus faecalis and Bacillus cereus. The effect of the combined compound is stronger than that of the separate constituents, and is as strong in the dark as in the light. Only 0.005% of the initial S. aureus population survive after a 2 h treatment. Absorption and fluorescence spectra of hemin-deuteroporphyrin mixtures in water and liposomes suggest the formation of a species with spectroscopic properties which are different from those of the two constituents.

In order to photosensitize Gram (-) bacteria such as Pseudomonas aeuruginosa and Escherichia coli, we introduced the small peptide polymyxin-B nona-peptide (PBNP) which stimulated the translocation of porphyrin through the outer membrane of these bacteria and makes PDT possible. Gram negative cell killing by the use of PBNP and DP broadens the antibacterial spectrum of photodynamic inactivation and opens new horizons for this modality as a wide spectrum drug when antibiotic resistance is the main concern. Plasmidial and chromosomal DNA damage in S. aureus and E. coli cells was mediated by DP photosensitization. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected and its degradation products were detected after treatment.

The unique antibacterial properties of Fe-protoporphyrin (haemin) on Staphylococcus aureus, compared to Co-protoporphyrin (Co-PP), Mg-protoporphyrin (Mg-PP) and Zn-protoporphyrin (Zn-PP) are described. Only haemin (20 microM) exhibits a strong light-independent antibacterial effect on S. aureus; the other metalloporphyrins, Co-PP, Mg-PP or Zn-PP, have no antibacterial effect in the dark. Only light photosensitization of Mg-PP-treated cells resulted in the inhibition of the bacterial growth, while Co-PP or Zn-PP were photodynamically inactive. A notable effect of haemin on inactivation of S. aureus was the induction of immediate ion fluxes as determined by X-ray microanalysis (XRMA) of fast-frozen cells. A marked efflux of K (96%) and Cl (94%) was expressed immediately as determined by X-ray microanalysis of S. aureus cells treated with haemin for 5 min. Only 48% loss of Na was detected in the cells under these treatment conditions, while P content was increased by 150%. Electron microscopy analysis revealed the appearance of a mesosome-like structure connected to the new septa, filamentous chromosome and arrays of aggregated ribosomes in the cytoplasm. We propose that haemin has multiple cellular targets for its oxidative effect in S. aureus.

bacterial and human leukemic cells were carried out with these sensitizers, in comparison with HPD andPhotofrin II. The new sensitizers were found to have high quantum yields for production of singlet oxygen insolutions. They also have comparable photosensitizing capacities of bacterial cells.1. INTRODUCTIONHematoporphyrin derivative (HPD) and its photoactive fraction Photofrin II (PF-II), have been used forseveral years in photodynamic therapy (PDT) of cancer.14 Their utilization as sensitizers relies on theirselective uptake and retention by cancerous cells,5'6 and on the light-activated cell damage, which is caused byeither an electron or hydrogen transfer from the triplet sensitizer to a substrate, (Type I mechanism), or bysinglet oxygen mediation (Type I1) For clinical applications, long-wavelength light is preferred for its deeperpenetration into the tissue. The light sources which are mainly being used are HeNe and dye lasers or filteredxe lamps. The photosensitization action spectra of Hpd and PF-II exhibit only marginal bands in the red,peaking around 630 nm.78 Therefore, high doses of HPD or PF-II have to be administered, with a resultinglight sensitivity of the whole body for extended periods. The search for new sensitizers with stronger lightabsorption at long wavelengths, which would enable the use of long-wavelength lasers as well as lowering theadministered doses, is currently being pursued.9"°

The mechanism of inactivation of Staphylococcas aureus cells by hemin is described. Protection experiments by sulfhydryl reagents such as cysteine, mercaptoethanol, glutathione or thioglycolate in their reduced form prevent S. aureus bacteria from inactivation by hemin (1.5 × 10−5 M). The treatment of bacteria by hemin in the presence of one of those reagents (1 × 10−2 M) showed that the growth rate and viability of the culture remained unchaged. On the other hand sulfhydryl reagents did not prevent the binding of hemin to the bacteria. When cysteine or glutathione were introduced to a culture after exposure to hemin it could neither reverse the damage done to the cells nor shorten the time of the culture's recovery. Another type of protection was obtained by addition of serum albumin which prevented hemin molecules from binding to the bacterial envelopes. Furthermore, when albumin was introduced after the bacteria were treated by hemin it prevented further damage to the survivors and thus shortened the time required for recovery. None of the singlet oxygen quenchers or hydroxyl radical scavengers could protect the bacteria from hemin inactivation. The mechanism by which hemin affects S. aureus is assumed to be by oxidizing a major system within the cell.

The porphyrin-dependent inactivation ofStaphyloccocus aureus and the induction of mesosomal structures are described. The antimicrobial activity of different photoactivated porphyrins was compared with the dark cytotoxic effect of hemin. Deuteroporphyrin, protoporphyrin, hematoporphyrin, and hematoporphyrin derivative (Hpd) markedly reduced cell growth upon irradiation with light. Photofrin II, the polymerized fraction of Hpd, and other synthetic porphyrins had no effect on staphylococcal growth. Hemin immediately inhibited cell viability in the dark and induced the development of an irregular cell wall, analyzed by scanning electron microscopy (SEM). Inside the cytoplasm a multivesicular mesosome was formed near the septum, as detected by transmission electron microscopy (TEM). The mesosomal volume and its frequency in the cells was increased in a time-dependent manner. The mesosome appearance was not related to fixation by glutaraldehyde or to post-fixation by osmium tetroxide. Glycosyl moieties stained by ruthenium red revealed the formation of membrane-like structures in the mesosome. It is concluded that oxygen-dependent reactions potentiated by porphyrins may induce disturbances in the synthesis of staphylococcal membrane and cell wall, revealed as mesosomes.

The antibacterial activity of hemin onStaphylococcus aureus is described. Hemin binding to bacteria was a rapid process, and each cell accumulated 5×105 to 1×106 molecules within 5 min. Bacterial growth was stopped completely after 30 min from addition of low concentration of hemin (3–10 μg/ml). Cell viability was reduced by 99.9% in 1 h of exposure, and the effect was consistent at any stage of the growth curve whenever hemin was added. Glucose utilization was arrested immediately after hemin addition, and no CO2 was produced. The survivors of hemin treatment regrow in a time-related kinetics depending on the dose of hemin to which the cells were exposed. The recovered bacteria were again sensitive to hemin, similar to an untreated culture. We suggested that the recovery phenomenon is a result of an “on-off” mechanism regulating sensitivity to hemin, rather than a selection mechanism giving rise to hemin-resistant mutants.