Comparative Photophysical Study of Disulfonated Aluminum Phthalocyanine in Unilamellar Vesicles and Leukemic K562 Cells

Abstract— The photophysical properties of cis -disulfonated aluminum phthalocyanine (AlPcS₂) in unilamellar vesicles (liposomes) of DL-a-dipalmitoyl-phosphatidylcholine have been measured. Both the fluorescence and triplet quantum yields decreased with increasing sensitizer concentration. The time-resolved fluorescence decays, analyzed by both the sum of exponentials and decay time distribution analyses, are compared with those reported for AlPcS₂ in leukemic K562 cells. Information on the pho-todynamic transport and localization mechanism has been obtained by drawing correlations between the two systems, indicating active transport of the phthalocyanine into tumor cells involving lysosomal accumulation.     

A Study of the Uptake of Toluidine Blue O by Porphyromonas gingivalis and the Mechanism of Lethal Photosensitization

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (³H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium-neon (HeNe) laser and TBO was investigated by using deuterium oxide (D₂O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L-tryptophan. There were 9.0 log₁₀ and 2 log₁₀ reductions in the presence of D₂O and H₂O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm²), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/ cm²) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggre-gation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.     

Comparison of distribution and photodynamic effects of di- and tetra-sulphonated aluminium phthalocyanines in normal rat colon

We have previously reported photodynamic therapy of normal rat colon using aluminium sulphonated phthalocyanine (AISPc). In that study, the AISPc used was a mixture of phthalocyanines of different degrees of sulphonation. Phthalocyanines of defined degrees of sulphonation have recently become available and we compared the distribution of the di- and tetra-sulphonates (AIS2Pc and AIS4Pc) in rat colon and colon wall structures employing both chemical extraction and fluorescence photometry using a charge coupled device imaging system. Also, the photodynamic effects produced by these components in rat colon were compared at various times after photosensitization. After intravenous photosensitizer administration using equimolar doses, the concentration of AIS2Pc in colon fell off more rapidly with time than AIS4Pc. Differences were noted in the microscopic distribution of these compounds, with the di-sulphonate exhibiting peak fluorescence in colon wall structures by 1 h after photosensitization, while mucosal fluorescence with the tetra-sulphonate peaked at 5 h. Fluorescence was also lost from the colon wall much more slowly with the tetra-sulphonate, which tended to be retained in the submucosa. Maximum photosensitizing capability was seen at 1 h with AIS2Pc and no lesions could be produced with photodynamic therapy at 1 week, with up to 5.65 mumol/kg. With AIS4Pc (5.65 mumol/kg), while no lesions could be produced with light treatment at 1 h, photodynamic therapy at 1 week produced lesions only slightly smaller than those produced with treatment at 48 h (the time of maximum effect), and significant photosensitization was present at 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the photodynamic effect of exogenous photoprotoporphyrin and protoporphyrin IX on PAM 212 murine keratinocytes

A comparative study of the cellular photosensitizing properties of protoporphyrin IX (PpIX) and photoprotoporphyrin (Ppp) was carried out in the transformed murine keratinocyte cell line, PAM 212. Time-course fluorescence studies were performed to determine the rate of uptake by cells together with fluorescence microscopy. The sensitized cells were laser irradiated with a range of light doses at 635 or 670 nm to determine the phototoxicity of the two compounds and to investigate their relative fluorescence photobleaching properties. Ppp showed enhanced phototoxicity at both its optimal activation wavelength of 670 nm (eight times more phototoxic than PpIX activated at its optimal wavelength of 635 nm for the same fluence) and at 635 nm (three times more phototoxic than PpIX at the same wavelength), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The photobleaching rate of Ppp in cells was found to be higher using 670 nm irradiation compared with that of PpIX at 635 nm irradiation. At 635 nm, however, the photobleaching rate of Ppp was comparable to that of PpIX. The photobleaching quantum yields of the two compounds in cells were found to be similar at approximately 5 x 10(-4), with the same value confirmed at both 670 and 635 nm irradiation for Ppp. The fluorescence lifetime of Ppp in cells was measured as 5.4 ns using time-correlated single photon counting.     

Effect of Dosimetric and Physiological Factors on the Lethal Photosensitization of Porphyromonas gingivalis in vitro

The aims of this study were to (1) determine the effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis using tolui-dine blue O (TBO) and light from a helium/neon (HeNe) laser; (2) determine the influence of sensitizer concentration, preirradiation time, serum and growth phase on sensitizer uptake by P. gingivalis. The dosimetric factors studied were concentration of TBO, light dose and preirradiation time. The physiological factors were presence of serum, pH and bacterial growth phase. Sensitizer uptake by P. gingivalis under various conditions was determined using tritiated TBO (³H-TBO). In the presence of TBO, a light dose-dependent increase in kill was attained (100% kill at 4.4 J). There was no significant effect on the numbers killed when TBO was increased from 12.5 to 50 µg/mL. An increase in preirradiation time gave slightly increased kills. High kills were achieved at all three pH (6.8–8.0). Although kills were substantial in the presence of serum, they were significantly less than those obtained in the presence of saline. Cells in all three growth phases were susceptible to lethal photosensitization, although stationary phase cells were slightly less susceptible. Maximum uptake of TBO occurred within 60 s and uptake in serum was less than in saline. The uptake by the log phase cells was greater at lower concentrations of sensitizer (50 µg/mL), compared to the other two phases.     

Fully Protected Glycosylated Zinc (II) Phthalocyanine Shows High Uptake and Photodynamic Cytotoxicity in MCF‐7 Cancer Cells

Phthalocyanine photosensitizers are effective in anticancer photodynamic therapy (PDT) but suffer from limited solubility, limited cellular uptake and limited selectivity for cancer cells. To improve these characteristics, we synthesized isopropylidene‐protected and partially deprotected tetra β‐glycosylated zinc (II) phthalocyanines and compared their uptake and accumulation kinetics, subcellular localization, in vitro photocytotoxicity and reactive oxygen species generation with those of disulfonated aluminum phthalocyanine. In MCF‐7 cancer cells, one of the compounds, zinc phthalocyanine {4}, demonstrated 10‐fold higher uptake, 5‐fold greater PDT‐induced cellular reactive oxygen species concentration and 2‐fold greater phototoxicity than equimolar (9 μm ) disulfonated aluminum phthalocyanine. Thus, isopropylidene‐protected β‐glycosylation of phthalocyanines provides a simple method of improving the efficacy of PDT.