Ivo Cozzani

Distribution of endogenous and injected porphyrins at the subcellular level in rat hepatocytes and in ascites hepatoma.

Different doses (0.5-20 mg/kg) of hematoporphyrin (HP) have been injected intraperitoneally into normal rats and rats affected by Yoshida ascites hepatoma. About 80% of HP reaching the liver was recovered in the extracellular compartment after liver perfusion, the ratio of extra- to intracellular HP being essentially independent of the administered dose. Similar data were obtained at different times after injection of 20 mg/kg HP. Intracellular HP largely accumulates in the mitochondria and in the membrane components of the nuclear fraction of isolated hepatocytes. Kinetic studies suggest that the cell receptors of highest affinity for HP are present in the external membrane. The latter result obtains for ascites hepatoma cells in an even more evident way, although the latter cells exhibit secondary HP binding sites probably constituted by cytoplasmatic proteins. Moreover, the clearance of intracellular HP from malignant cells occurs at a remarkably lower rate as compared with HP clearance from liver cells.

Efficient photosensitization of malignant human cells in vitro by liposome-bound porphyrins

Comparative kinetics of porphyrin uptake and release by HeLa cells, incubated with equivalent concentrations of either hematoporphyrin (Hp) in aqueous solution or Hp and its dimethylester (HpDME) bound to unilamellar liposomes, show that liposomal porphyrins are bound at a higher rate and in considerably larger amounts. Moreover, the release of cell-bound porphyrins into the medium is remarkably reduced and slowered after cell loading with liposome-bound porphyrins. The presence of 1% bovine or human serum albumin (but not serum globulins) in the medium has no effect on uptake and release of liposome-bound porphyrins by HeLa cells, whereas it remarkably decreases the uptake of aqueous Hp. Parallel studies of cell photodamages under known concentrations of cell-bound porphyrin unequivocally demonstrate that the photodynamic effect is strictly related to the porphyrin load. As a consequence a dramatic increase of cell-photosensitizing efficiency is obtained by binding Hp (and even more HpDME) with liposomes. Electron microscopy investigations on cell damages caused by loading with liposome-bound porphyrins and subsequent illumination show that the plasmatic membrane is one important cell site of porphyrin interaction and photodynamic effect.

Interaction of Free and Liposome-Bound Porphyrins with Normal and Malignant Cells: Biochemical and Photosensitization Studies in Vitro and in Vivo

The porphyrins used in combination with visible light for the early diagnosis and phototherapy of neoplasias are so far limited to hematoporphyrin (Hp) alone or associated with some derivatives obtained by acid treatment (HpD). Several evidences indicate that the cytoplasmic membrane represents a hydrophobic barrier limiting the uptake of HpD and, to a greater extent, of Hp by cells; in most cases, a significant aliquot of cell-bound porphyrin is rapidly cleared from both isolated cells1 and animal tissue2. The release is markedly slower for malignant cells and tissues3 thus providing favourable tumor-to-normal tissue ratios of porphyrin concentrations, which are exploited in diagnostic and therapeutic oncology. Clearly, further enhancements of these ratios would be of paramount clinical importance. In this paper, we report some experimental indications that liposome-bound hematoporphyrin (Hplip) and its water-insoluble dimethylester (HpDME) are taken up by cells and tissues at a higher rate and in greater amounts as compared with aqueous Hp solutions (Hpaq) of corresponding concentrations. Moreover, the release from the cells of porphyrins vehiculated via liposomes is slower.

Molecular and Cellular Mechanisms in Photomedicine: Porphyrins in Microheterogeneous Environments

Photophysical and photokinetic studies on porphyrin-sensitized photoreactions in homogeneous solutions indicate that the mechanism and efficiency of the photoprocesses are influenced by several factors, out of which the nature of the reaction medium plays a major role. Now, the tissue distribution and subcellular localization of porphyrins in vivo is related to their physico-chemical characteristics, concentration, aggregation state and affinity for specific biomolecules (e.g., serum proteins); hence, one would expect that a large variety of microenvironments are possible. Moreover, the heterogeneous nature of cells originates different mutual situations between the porphyrin photosensitizer and the potential targets, depending on their distance, levels of motility, separation by interphases, etc. The oxygen concentration and the reactivity of the transient species can also vary considerably in different subcellular districts.

Monitoring of hematoporphyrin injected in humans and clinical prospects of its use in gynecologic oncology.

The use of hematoporphyrin (HP) and its water soluble derivatives (HPD) as tumor localizers1 and sensitizers for phototherapy of neoplasias 2 is well documented and receiving rapidly increasing interest.

Separation and characterization of NAD- and NADP-specific succinate-semialdehyde dehydrogenase from Escherichia coli K-12 3300

Two distinct proteins endowed with succinate-semialdehyde dehydrogenase (succinate-semialdehyde:NAD(P)+oxidoreductase, EC 1.2.1.16) activity were separated and partially purified by ammonium sulphate fractionation or Sephadex G-200 gel-filtration or both. They differ by coenzyme specificity (NAD or NADP), molecular weight, temperature and pH resistance, pH-activity curves, beta-mercaptoethanol activation. Moreover, the NADP-specific enzyme catalyzes only the oxidation of succinate-semialdehyde among a number of aldehydes tested, whereas the NAD-specific form is active also towards n-butyraldehyde. The Km for the substrate is also appreciably different according to the coenzyme specificity, while the Km values for NAD and NADP are quite similar. Finally, the growth of the cells on gamma-aminobutyrate as the sole source of nitrogen resulted in enhanced level of the NAD-dependent succinate-semialdehyde dehydrogenase, with concurrent decrease of the alternate enzyme activity. On the basis of the above results, distinct metabolic roles are suggested for the two enzymes forms.

Photo-oxidation of l-glutamate decarboxylase from Escherichia coli, sensitized by the coenzyme pyridoxal phosphate and by proflavin

Irradiation of L-glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from Escherichia coli by visible light absorbed by the intrinsic chromophore, pyridoxal phosphate, caused the selective modification of two methionines per enzyme monomer. The disulfoxide derivative exhibited modified circular dichroism, chromatographic and kinetic properties, suggesting a conformational role for the two methionine residues. Irradiation of the enzyme in the presence of proflavin revealed the presence of two distinct groups of tryptophan residues with markedly different photooxidation rate constants. No evidence of involvement of tryptophans in the catalytic mechanisms of the enzyme was obtained. The results are compared with those obtained on irradiation of L-glutamate decarboxylase from Clostridium perfringens.

Molecular and Cellular Mechanisms in Photomedicine: Porphyrins in Cancer Treatment

The use of visible light in combination with a photosensitizing dye for the treatment of various types of tumors has been repeatedly attempted since the early 1900s when topically applied eosin and sunlight were shown to induce the regression of skin tumors1. This approach appears to be particularly attractive since i) several photosensitizers with a polycyclic chemical structure, including xanthenes2, acridines3, and psoralens4, display a preferential affinity for neoplastic as compared with normal cells; and ii) most cell constituents, in particular proteins and nucleic acids, are insensitive to the direct action of visible light. In principle, these two properties should allow one to restrict the photodynamic effects causing cell inactivation and necrosis to the dye-loaded tissue, with no concomitant damage to normal dye-free tissues, provided a suitable set of light wavelengths is selected for irradiation. Thus, photodynamic therapy exhibits a potential advantage with respect to other therapeutic modalities for tumors which are based on the use of ionizing radiations; in the latter cases, the simultaneous irreversible damage of normal tissues often limits the extent and efficacy of the treatment. Toward this aim, an ideal photosensitizer should have the following properties: i) lack of systemic toxicity; ii) selective uptake and retention by malignant tissues; iii) selective absorption of the incident light; and iv) efficient generation of photoreactive species leading to destruction of malignant tissues.