EVIDENCE AGAINST THE PRODUCTION OF SUPEROXIDE BY PHOTOIRRADIATION OF HEMATOPORPHYRIN DERIVATIVE

Abstract Experiments were performed to ascertain whether superoxide anion (O₂⁻) was produced by the photodynamic activation of hematoporphyrin derivative (HPD). Three different systems were utilized to detect formation of O₂⁻, oxidation of epinephrine to adrenochrome, reduction of cytochrome c and reduction of nitro blue tetrazolium (NBT). The effects on these detectors under identical conditions for HPD + h ν were compared to those obtained with two O₂⁻ generating systems, riboflavin + by and xanthine-xanthine oxidase, and to a singlet oxygen generating system, photoradiation of methylene blue. The results indicated that HPD + hv differed from the two O₂⁻ generating systems in failing to reduce cytochrome c or NET, and that HPD + h ν was similar to the behavior of methylene blue + h ν . In addition, HPD + h ν but not the O₂⁻ generating systems could inhibit mitochondrial cytochrome c oxidase activity. We conclude that the photodynamic activation of HPD does not produce O₂⁻ as a major oxygen radical and that the effects of HPD + h ν on mitochondrial cytochrome c oxidase are not caused by O₂⁻.     

EFFICACY OF PHOTODYNAMIC THERAPY ON ORIGINAL AND RECURRENT RAT MAMMARY TUMORS

Abstract— Photodynamic therapy has demonstrated efficacy toward primary, metastatic and recurrent human tumors. Here, we investigated the ability of photodynamic therapy, using Photofrin, to inhibit growth of R3230AC mammary adenocarcinomas when tumors were treated as original implants and again as lesions recurring at the initial treatment site. The results demonstrate that both initial implants and lesions recurring after the first photodynamic treatment respond similarly to the same photodynamic therapy protocol, with mean tumor volume doubling times of ˜ 11 days in both cases. Cells cultured from original tumor implants or tumors that recurred after photodynamic treatment accumulate equivalent amounts of [¹⁴C]polyhematoporphyrin. Single cell suspensions prepared from either original or recurrent tumors from animals administered 5 mg/kg Photofrin and exposed to light in vitro displayed comparable phototoxicity. Additionally, examination of tumors by light microscopy revealed no morphological differences between the original tumor implants and the recurrent lesions. Taken together, these data indicate that lesions which recurred at the site of the initial photodynamic treatment were not resistant to a second identical course of photodynamic therapy.     

INHIBITION OF THE ATPase ACTIVITY OF P-GLYCOPROTEIN BY PORPHYRIN PHOTOSENSITIZATION OF MULTIDRUG-RESISTANT CELLS in vitro

Abstract— The effectiveness of photodynamic therapy against P-glycoprotein ATPase activity in multidrug-resistant cells was studied. Chinese hamster ovary AUXB1 (drug-sensitive) and CR1R12 (multidrug-resistant) cell lines were compared with respect to uptake of ¹⁴C-polyhematoporphyrin and porphyrin photosensitization. Phototoxicity of Photofrin® was similar in both cell lines, and no major differences in uptake or efflux of ¹⁴C-polyhematoporphyrin were observed. Porphyrin photosensitization in vitro of CR1R12 cells or isolated plasma membranes from these cells caused inhibition of P-glycoprotein ATPase activity. Application of porphyrin photosensitization at a sublethal level to CR1R12 cells resulted in a small but significant increase in adriamycin-induced cytotoxicity. The hydrophobic "picket-fence" porphyrin, meso -tetrakis-( o -propionamidophenyl)porphyrin,α,α,α,β-isomer, was more inhibitory toward P-glycoprotein ATPase activity than the two less hydrophobic porphyrins tetraphenylporphine tetrasulfonate and Photofrin®.     

Insulin binding and glucose transport in the R3230AC mammary adenocarcinoma.

Cells dissociated from the R3230AC mammary adenocarcinoma from intact and diabetic rats were examined for insulin binding and glucose transport. The Kd for insulin binding, approximately 10(--10) M, was similar in all tumors studied. However, the apparent number of receptor sites per cell increased in cells from diabetic rats. Kinetic analysis of 3-0-methyl glucose (3-OMG) entry showed both diffusional and passive carrier characteristics. Insulin (4 X 10(--9) M) in vitro did not affect diffusional entry, whereas the hormone altered the passive carrier system, as reflected by an increase in Km and Vmax. Insulin decreased initial velocity of glucose transport at 4--6 mM glucose levels but increased initial velocity of glucose transport at 20 mM glucose. An explanation of the role of insulin on tumor growth in vivo from effects on glucose transport in vitro is proposed.     

PICOSECOND FLUORESCENCE OF R3230AC MAMMARY CARCINOMA MITOCHONDRIA AFTER TREATMENT WITH HEMATOPORPHYRIN DERIVATIVE and PHOTOFRIN® II in vivo

Hematoporphyrin derivative (HPD) and other porphyrin samples were excited by 20-ps 532-nm laser pulses. Fluorescence was detected using a low-jitter streak camera. Data were fitted to a sum of exponential decay times on the order of picoseconds. Fluorescence of porphyrins in aqueous solution show various behaviors depending on the hydrophobicity of the porphyrins. The most hydrophilic porphyrins show long decays only (greater than 500 ps). Porphyrins intermediate in hydrophobicity have intensity-dependent fast decays. The most hydrophobic have fast decays (less than 20 ps). Picosecond fluorescences of mitochondria prepared from rat tumors treated in vivo with HPD or Photofrin II show an increase in the ratio of fast to slow decays when compared to the injected porphyrins. These results are consistent with the concentration of the more hydrophobic porphyrins in mitochondria in photosensitization treatment. Thus picosecond fluorescence studies of porphyrins may provide a means to obtain photoproperties which differentiate between effective and ineffective in vivo photosensitizers.     

Janus micelles induced by olefin metathesis

A facile one-step procedure for hydrophobic modification and simultaneous TEM contrast enhancement via a regioselective olefin metathesis reaction using Grubbs' catalyst is presented. Polyether diblock copolymers were investigated, and both the chain ends of the hydrophilic and the hydrophobic block were hydrophobically modified. Modification of the hydrophilic block results in nonsymmetric supramolecular structures (Janus micelles) which self-assemble into larger hierarchically organized super-micelles.     

Synthetic porphyrins at interfaces; photosensitization and related reactions of atropisomers of o-substituted tetraphenylporphine derivatives in films, reversed micelles and membranes

Abstract— Previous studies from our laboratories and elsewhere have shown that amides between fatty acids and the synthetic α, α, α, α -tetra (o-aminophenyl)-porphyrins exhibit good surfactant properties which facilitate their incorporation into structured assemblies characterized by hydrophobic-hydrophilic compartmentalization. This paper will focus on a number of aspects of our studies of these porphyrins at different interfaces. The α, α, α, α (4,0) isomers are readily incorporated into Langmuir-Blodgett films as either free base or metal complexes. Studies of assemblies containing free base and palladium (II) complexes have been carried out in which the porphyrin is irradiated in the presence of oxygen and nonexcited but oxidizable substrates. Much of the reactivity observed can be attributed to ¹O₂* generation. These studies reveal the migration range and reactivity of activated oxygen in a structure related to biomem-branes. Several of these synthetic porphyrins have also been examined in cell suspensions and in synthetic reversed micelle solutions. Studies in the former have shown that the porphyrins can mediate the photoinactivation of several enzymes located inside and within the mitochondrial membrane in tumor cells extracted from rats. They are found to compare favorably to hematoporphyrin derivative in effectiveness. Studies of the same porphyrins in their reactivity towards copper ion incorporation in anionic reversed micelles indicate striking rate differences which can be interpreted in part to structural variations between the porphyrins as well as to their orientation at the hydrophobic-hydrophilic interface.     

Quantum mechanics and phase transitions in small noble-gas clusters

The quantum-statistical properties of Argon clusters are calculated applying a Path-Integral Monte-Carlo (PIMC) method. Results for pair correlation functions and kinetic-and configurational energy expectations for various cluster sizes and temperatures are presented. Physical phenomena such as ground-state kinetic energy and quantum admixture of different atomic configurations are studied and experiences with the method are given. In particular, the relation between metastable cluster states and phase transitions is discussed.     

The long-time behaviour of the electric-field autocorrelation function in a finite photon gas

We investigate an autocorrelation function of a soluble three-dimensional system, namely the temporal coherence functionC _E(t)∝<E(0)E(t)> of the thermal radiation field in a cube-shaped cavity for the stochastic electrical fieldE. In the thermodynamic limit,C _E(t) relaxes exponentially at intermediate times, but a “long-tail” behaviourC ₀(t)=At^?4 withA<0 is predominant for long times. In the case of a finite, but not too small, cavity lengthL obeyingΛ=hc/k _BT?L and at timest withct?L, C _E(t) is described by an asymptotic expansion in powers ofL ^?1 using generalized Riemann zeta functions. Surface-and shape-effects enhance the long-tail. In the case of very small cavities withL«Λ, we calculate an expansion ofC _E(t) in terms of exp(?L ^?1) and cosines. An oscillatory, but not strictly periodic, long-time behaviour is observed in this case.