Varda Gottfried

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.

Photosensitizers in organized media: singlet oxygen production and spectral properties

Abstract— Measurement of singlet oxygen production by porphyrin‐type photosensitizers in simple buffer solutions show that TPPS is more efficient than PII (Photofrin II) or than hematoporphyrin. This behavior was observed using three independent analytical detection methods for 1O2: RNO bleaching, tryptophan degradation, and oxygen consumption. Similar results were obtained when irradiating with a classical light source or with a pulsed dye laser. Addition of EPC liposomes to the buffer solution caused a decrease of efficiency for TPPS and an increase for PII. Addition of BSA shows the same relative pattern: a small increase of efficiency for TPPS and a tenfold increase for PII. These changes can be ascribed to specific interactions between the sensitizer molecules and the organized medium. Since changes in the fluorescence properties are also due to interactions with the medium, we monitored the emission of the sensitizers as a function of the environment. The fluorescence peak positions (at 648, 705 nm for TPPS and at 615, 677 nm for PII) were all red shifted. The intensities show an increase, particularly for PII in liposomes, due to a marked change in the microviscosity.

In vivo damage to chorioallantoic membrane blood vessels by porphycene-induced photodynamic therapy☆

Photodynamic therapy (PDT) was performed in the chick embryo chorioallantoic membrane (CAM) for the purpose of quantitative evaluation of several porphycenes as potential photosensitizers. Porphycenes are structural isomers of porphine possessing lower symmetry of the macrocycle and are characterized by 10-fold higher absorption at the therapeutic wavelengths for PDT (lambda > 630 nm). PDT-induced damage to CAM blood vessels included vasoconstriction and blanching as was monitored during irradiation and videotaped. Image analysis techniques enabled us to follow PDT-induced constriction of vessel diameter (to 50%), reduction of blood perfusion (to 40% lower optical density) and shrinkage of implanted tumours (to 10% of their original area). The observed PDT efficacy of functionalized porphycenes is positively correlated with the number of polar substituents.

PHOTODYNAMIC EFFICACY OF NATURALLY OCCURRING PORPHYRINS IN ENDOTHELIAL CELLS IN VITRO AND MICROVASCULATURE IN VIVO

Photodynamic therapy (PDT) has been described in terms of cellular and vascular effects. The precise mechanisms of cellular and vascular damage are still unknown. In this study, the photodynamic inactivation of endothelial cells in vitro and damage to the microvasculature in vivo by naturally occurring porphyrins (uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP)) were investigated. The chick chorioallantoic membrane model (CAM model) was used, which is convenient for the study of damage to the microcirculation induced by PDT. The hydrophilic porphyrins UP and CP exhibited low cytotoxicity towards endothelial cells. Only small amounts of UP and CP were taken up, resulting in weak inactivation after irradiation. In contrast, the more lipophilic PP showed a marked cytotoxicity. Considerable amounts of PP were accumulated in the cells, leading to pronounced inactivation after light exposure. For the three porphyrins, damage to the microvasculature was observed. The damage caused by the hydrophilic porphyrins UP and CP was strongly dependent on the drug and light dose. For vascular injury, the efficacy was graded as UP < CP < PP.

Temperature effects on photosensitized processes

The singlet-oxygen-mediated reaction of meso-tetraphenylporphine tetrasulphonate (TPPS4) with different chemical acceptors in buffered aqueous solution was studied as a function of temperature. Imidazole, tryptophan, dimethyl p-nitrosoaniline, (RNO) and furfuryl alcohol served as acceptors. The measurements were performed in real time by spectroscopic or electrochemical monitoring of the consumption of the various reagents, acceptors or dissolved oxygen as a function of the absorbed energy. The results show the following increases in the reaction rate over the temperature range 15-45 degrees C: tryptophan (86%), RNO (90%), furfuryl alcohol (150%) and imidazole (210%). The influence of temperature-correlated changes in the initial oxygen concentration and pH was investigated. Possible implications of the present results for the synergistic influence of hyperthermia and photodynamic therapy are discussed.

Photodynamic activity of porphines on tubulin assembly

Porphyrins and porphine analogs have been shown to induce cytotoxic effects on cells and tissues after exposure to light, an effect which is currently being studied as a new modality for treatment of cancer, termed photodynamic therapy (PDT). One of the important factors in PDT is the preferential uptake of sensitizers by rapidly proliferating tissues. Previous studies showed that cytoskeletal structures are affected by porphyrin-induced PDT. In the present study we investigate the inhibitory efficiency of porphines on tubulin assembly in vitro. We analyze the efficiency of several sulfonated porphine isomers: tetraphenylporphine n-sulfonate (TPPSn) where n equals 4, 2a and 2o (a and o refer to adjacent and opposite substitution, respectively) and the structural isomers of tetra(o-,m-, and p-hydroxyphenyl)porphine (o-,m- ,p-THPP), in order to find a possible structure-activity relationship. The efficiency of the sensitizers was assayed by their capacity to inhibit microtubule assembly. Binding to monomeric tubulin is essential for effective inhibition of assembly, with or without exposure to light. Without exposure to light, TPPS2o was found to be the most potent inhibitor, followed by TPPS2a and to a much smaller extent by TPPS4. All THPP isomers have negligible inhibitory effect. Upon exposure to white light, microtubule assembly was inhibited in the same order:TPPS2o greater than TPPS2a greater than TPPS4 greater than THPP. All porphines were found to have high affinity to the same site on tubulin even those who had almost no dark effect on tubulin assembly (THPP). Addition of the porphines to assembled microtubules did not lead to their depolymerization even after prolonged irradiation. Since it was previously suggested that porphines may share the same binding site on tubulin as bis-ANS, a known tubulin assembly inhibitor, we performed competition studies with this inhibitor and the porphines. It was shown that bis-ANS does not share the same site on tubulin as the porphines and therefore their effects are additive.

Uptake and photodynamic activity of porphycenes in tumor cells implanted on the chick chorioallantoic membrane (CAM)

The chick chorioallantoic membrane (CAM) is a convenient model for the study of photodynamic therapy (PDT). This membrane has a rich vasculature, which mimics the tumor neovasculature, and can also serve as a host for implanted tumors. The transparency of the CAM enables in-vivo monitoring of vascular changes during and post PDT, without the need to sacrifice test animals at each time point. Video documentation and analysis of events occurring during and after irradiation permit the quantification of changes in vessel morphology, blood perfusion and tumor development. The compounds tested in this study belong to a family of potential sensitizers -- the porphycenes. These are phorphyrin isomers based on a 16-membered macrocycle, in which the four methine moieties linking the pyrrole rings have been replaced by two direct bonds and two ethine bridges. Experiments were performed on blood vessels of the intact CAM and on recurrent human melanoma cells implanted on the CAM. Tumor selectivity was demonstrated by measuring drug uptake using fluorescence methods. A sensitizer injected systemically into the embryo yolk sac could be detected in the blood vessels 30 min after injection; 1 h later the sensitizer had preferentially accumulated in the tumor. Tumors were irradiated at the optimal uptake time (after 1 h) for 16 min with a 20 mW HeNe laser. Video image analysis showed that 96 h after irradiation tumors had decreased to 5% of their original size. In contrast, non-irradiated control tumors on the same CAM, continued to proliferate and grew to more than twice their original size. In addition, we observed a difference in the damage mechanism after systemic compared to topical administration. Topical application followed by irradiation caused fast necrosis of tumors, which might suggest direct damage to tumor cells, whereas after systemic administration, PDT damage was manifested by slower necrosis, presumably caused by vascular destruction.

Real-time fluorescence microscopy monitoring of porphyrin biodistribution

In vivo uptake of the natural porphyrins, uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), was monitored by fluorescence microscopy. Experiments were performed using the chick chorioallantoic membrane (CAM) model, which allowed video documentation of fluorescence both in real time and after integration over a chosen time interval (usually 2 s). Sensitizers at a concentration of 50 (mu) M (100 (mu) L) were injected into a medium-sized vein (diameter approximately 40 micrometer) using an ultra-fine 10 micrometer diameter needle. Fluorescence images were quantitated by subtracting the fluorescence intensity of surrounding CAM tissue (Fmatrix) from the intravascular fluorescence intensity (Fintravascular), after transformation of the video frames into digital form. The differential fluorescence intensity, Fintravascular - Fmatrix, is a measure of the biodistribution. Real time measurements clearly showed that CP and UP fluorescence is associated with moving erythrocytes and not with endothelial cells of the vessel wall. Fluorescence intensity was monitored, up to 60 minutes after injection, by averaging the fluorescence over time intervals of 2 s and recording the integrated images. The fluorescence intensity reached its maximum in about 20 - 30 min after injection, presumably after monomerization inside erythrocyte membranes. The results are interpreted in terms of physical-chemical characteristics (e.g. hydrophilicity) and correlated with the photodynamically induced hemostasis in CAM blood vessels.

Laser photodynamic therapy of cancer: the chorioallantoic membrane model for measuring damage to blood vessels in-vivo

The mechanism of photodynamic therapy (PDT) involves, as a primary step, damage either directly to the tumor cells or to the surrounding vasculature which, in turn, causes disruption of tumor blood flow and, ultimately, tissue necrosis by anoxia. We report here a novel in-vivo model for investigating vascular events during PDT. The chick chorioallantoic membrane (CAM) model was chosen since it is an established model for studying biological processes such as implantation and angiogenesis . The photosensitizers meso-tetraphenylporphine tetrasulfonate (TPPSJ and chloro-aluminum phthalocyanine tetrasulfonate (CAPcS) were topically applied onto the CAM. In all cases where sensitizer plus radiation was administered, changes in the CAM microcirculation occurred, as viewed through a stereoscopic microscope. With increasing light/drug dose we observed capillary leakage, stasis, occlusion, hemorrhage, and engorgement. The course of damage formation was documented in real time by video photography. All controls (sensitizer alone or light alone) remained unchanged compared to treated CAM. This work also describes preliminary experiments on tumor cells transplanted onto the CAM.

Video microscopy of vascular damage during photodynamic therapy

Photodynamic therapy (PDT) was performed in the chorioallantoic membrane (CAM) of the chick embryo. This is a convenient model to study vascular effects. PDT-induced damage was monitored continuously during irradiation and recorded on a VCR driven by a PC. Time-lapse video documentation provided a detailed view of the entire process: tumor growth, angiogenesis, vascularization, PDT, and tumor regression. Our particular interest was in resolving the controversy regarding the primary mechanism in PDT, as to whether the predominant damage is to the parenchymal tumor cells or to the vascular endothelium. Image analysis techniques enabled us to follow quantitatively changes occurring during the process. These changes included alternations in blood vessels (color and morphology) and in tumors (tumor area).