PHOTOSENSITIZED PROCESSES BY TETRAPYRROLIC DERIVATIVES

PHOTOSENSITIZED PROCESSES BY TETRAPYRROLIC DERIVATIVES: APPLICATIONS FOR THE DEVELOPMENT OF PHOTOTERAPEUTICAL TECHNIQUES.

Principal Investigator: Dr. Fernanda Ricchelli

Staff: Silvano Gobbo, Giuseppe Tognon

Fernanda Ricchelli

External collaborations:
Prof. Giulio Jori, Dr Elena Reddi; Department of Biology, Padova.
Prof. Paolo Bernardi, Dr Valeria Petronilli, Dr Fabio di Lisa; Department of Biomedical Sciences, Padova.
Dr. Federica Dabbeni-Sala, Dr Sergio Bova; Department of Pharmacology, Padova.
Dr. Christian Salet and Giuliana Moreno Laboratoires de Biophysique et de Photobiologie, INSERM et CNRS, Muséum National d'Histoire Naturelle.
Dr. Peter Nikolov; Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Prof. M. Brimble and Dr. T. Muir, Department of Chemistry, University of Auckland, New Zealand.

Introduction

This research project was aimed at the understanding of the mechanisms of photosensitization, i.e. the process induced by highly toxic, reactive oxygen species (ROS), produced after irradiation by UV-visible light of some fluorescent molecules (in particular, porphyrins and analogous compounds, such as phthalocyanines, naphthalocyanines, porphycenes etc.) that is involved in some types of cell damage and death.

The photosensitization process can be highly noxious and cause irreversible cell damages, as observed in human porphyria or in skin lesions such as erythema and cancer. However, porphyrins and other photosensitizers have found useful biomedical applications in the treatment of several deseases, as, for ex., in the photodynamic therapy of tumors (PDT) and in the destruction of atheromatous plaques and psoriatic lesions. Pioneering studies concern the use of photosensisting molecules as antibacterial and antiviral agents.

Most porphyrins which have been tested both in vitro and in vivo carry on their photoactivity after preferential association to membranes (in particular, plasma, mitochondria and endoplasmic reticulum membranes), and the targets of photodamages are proteins and lipids. The extent of photodamage mainly depends on the sensitizer aggregation state (only monomeric species are photoactive) and its distribution pattern into cells and subcellular organelles. The maximum photoprocess efficiency, in fact, is reached when photosensitizer and target are localized in close proximity, being the diffusion path of the oxygen cytotoxic species strictly limited (< 0.1µm in biological material) owing to their high reactivity.

This research was focused on the characterization of the photochemical and photophysical properties of photosensitizing agents in model and biological membranes as well as on the study of their uptake and photodamage mechanisms on functional activities of cells and subcellular organelles. Particularly studied was the correlation between the distribution properties of porphyrins and derivatives and their photosensitizing efficiency in mitochondria, which are primary targets of the photodynamic effect in vivo. It was shown that the distribution pattern of these molecules in membranes is influenced by several factors including the degree of dye hydrophobicity, the nature of the carrier and the incubation time with the membrane. The transport system of photosensitizers in vivo is a crucial determinant. A primary goal in the photodynamic treatment of tumours, for ex., concerns the achievement of tumor selective targeting by the photosensitizer. To this aim, most researches were oriented to obtain the specific release of the drug to the low density lipoproteins (LDL). It has been credibly hypothesized that, in vivo, LDL play the role of preferential carriers of the sensitizer to tumoral tissues. In fact, LDL interact with several types of tissues, in particular hyperproliferating tissues, via an endocytosis-mediated process. In agreement, LDL-associated photosensitizers administered to tumor-bearing rats accumulate preferentially in malignant tissues as compared to the drugs freely dissolved in aqueous solutions or incorporated in other lipoproteins lacking of specific receptors on plasma membranes.

Recently, this research has been focused on the effects of the photosensitization-mediated oxidative stress on the permeability transition (PT) process of the mitochondrial inner membrane, that has been postulated to be involved as early step in the cascade of events leading to apoptosis.

Projects underway

1. Effects of photosensitizing agents on structure and function of cells and subcellular organelles.

This project is aimed at the control of distribution pattern of photosensitizers in vivo. This can result in a high selectivity of the photoinduced damage (normally limited to the close surroundings of the sensitizing agent). Among cellular targets, mitochondria are of particular interest because of the key role performed by these organelles in cell metabolism as well as in the regulation of normal cell functions. The main research lines are:

a) selection of photosensitisers specifically targeted to mitochondria which may provide versatile applications in studies of mitochondrial contribution to processes leading to cell death. Cationic porphyrin derivatives with specific chemical structure are under investigation.

b) Identification of the ROS generated by the selected photosensitizers. It is known that the photoinduced damage in biomolecules can occur via two general mechanisms: direct damage of biological targets where an electron transfer event is the initial step (Type I); singlet oxygen (¹O₂) production where an energy transfer reaction from the photoexcited molecule to molecular oxygen is the initial step (Type II). Recent literature data indicate that the competition between electron and energy transfer in the reaction site affects the ratio of apoptosis versus necrosis cell death.

c) Effects of the photosensitization induced-oxidative stress on the mitochondrial permeability transition (PT), a process involved in the early steps of apoptosis.

2. Generation of ROS by photoactivated cell-probes and implications for studies of cellular functions.

Photochemical effects are undesirable when fluorescent probes are used for studies of cell viability and microscopic imaging. Microscopic observations using epifluorescence or laser illumination in confocal microscopy may result in the perturbation of cellular structures and induction of severe cell damage if dye concentrations and exposure times to the light are not properly tested. Actually, because of their phototoxic effects, some potentiometric fluorescent probes are under investigation as potential therapeutic agents against various types of tumors, which appear to accumulate these molecules more efficiently than normal, non transformed cells. A major goal of this research is to thoroughly characterize some of the dyes widely used in cell studies for phototoxicity and for direct effects on cell functions, and to define experimental conditions that allow their safe use with living cells. In particular, the attention is focused on Rhodamine and Rhodamine derivatives, including tetramethylrhodamine methyl ester (TMRM) which exclusively stains mitochondria and is at the present the most used probe for measurements of membrane potential.

PUBLICATIONS 1994-2001

INTERNATIONAL FULL PAPERS

1. DELLINGER, M., RICCHELLI, F., MORENO, G. & SALET, C. ( 1994 ) Hematoporphyrin Derivative ( Photofrin ) Photodynamic Action on Ca2+ Transport in Monkey Kidney Cells ( CV-1 ). Photochem. Photobiol., 60 : 368 - 372

2. RICCHELLI, F., NIKOLOV, P., GOBBO, S., JORI, G., MORENO, G., SALET, C. ( 1994 ) Interaction of phthalocyanines with lipid membranes : a spectroscopic and functional study on isolated rat liver mitochondria . Biochim. Biophys. Acta , 1196 : 165 - 171. I.F. 2.43

3. RICCHELLI, F. & GOBBO, S. (1995) Porphyrins as fluorescent probes for monitoring phase transitions of lipid domains in biological membranes. Factors influencing the microenvironment of hematoporphyrin and protoporphyrin in liposomes. J. Photochem. Photobiol. B : Biol., 29 : 65 - 70

4. RICCHELLI, F. (1995) Photophysical properties of porphyrins in biological membranes . J. Photochem. Photobiol. B : Biol., 29 : 109 - 118

5. RICCHELLI, F., GOBBO, S., JORI, G., MORENO, G &. SALET, C. (1995) Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes 1. The partition of hematoporphyrin and protoporphyrin in liposomes . Eur. J. Biochem., 233 : 159 - 164

6. RICCHELLI, F., GOBBO, S., JORI, G., SALET, C. & MORENO , G. (1995) Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes. 2. The partition of hematoporphyrin and protoporphyrin in mitochondria. Eur. J. Biochem., 233 : 165 - 170

7. SHOPOVA, M., STOICHKOVA, N., MILEV, A. PEEV, M., GEORGIEV, K., GIZBREHT, A., JORI, G. & RICCHELLI, F. (1995 ) Photodynamic Therapy of Experimental Tumours with Zn(II)-phthalocyanine and Pulsed Laser Irradiation. Lasers .Med. Sci., 10 : 43 - 46

8. MICHAILOV, N., PEEVA, M., ANGELOV, I., WOHRLE,D., MULLER, S., JORI, G, RICCHELLI, F. & SHOPOVA, M. (1997) Fluence Rate Effects on Photodynamic Therapy of B16 Pigmented Melanoma J. Photochem. Photobiol. B : Biol. , 37 : 154-157

9. SALET,C., MORENO,G. & RICCHELLI, F. (1997) Effects of PhotofrinÒ Photodynamic Action on Mitochondrial Respiration and Superoxide Radical Generation Free Radical Res. , 26 : 201-208

10. SALET, C., MORENO, G. , RICCHELLI, F. & BERNARDI, P. (1997) Singlet oxygen produced by photodynamic action causes inactivation of the mitochondrial permeability transition pore J. Biol. Chem., 272, n° 35 : 21938-21943

11. MANTAREVA, V., SHOPOVA, M., SPASSOVA , G., WÖHRLE D., MULLER, S., JORI, G. & RICCHELLI, F. (1997) Si(IV)-Methoxyethylene-glycol-naphthalocyanine: Synthesis and pharmacokinetic and photosensitizing properties in different tumour models J. Photochem. Photobiol. B : Biol. , 40 : 263-272

12. RICCHELLI, F., GOBBO, S., MORENO, G., SALET, C., BRANCALEON, L. and MAZZINI, A. (1998) Photophysical properties of porphyrin planar aggregates in liposomes Eur. J. Biochem., 253 : 760-765

13. SALET, C., MORENO, G. and RICCHELLI, F. (1998) Effects of photodynamic action on respiration in non-phosphorylating mitochondria Arch. Biochem. Biophys. , 358 : 257-263

14. RICCHELLI, F., BARBATO, P., MILANI, M., GOBBO, S., SALET, C. and MORENO, G.
Porphyrin photodynamic action on the Ca²⁺ influx in endoplasmic reticulum. .A comparison with mitochondria. Biochem. J. 338 : 221-227 (1999)

15. WÖHRLE, D. MÜLLER, S. SHOPOVA, M., MANTAREVA, V., SPASSOVA, G., RICCHELLI, F. and JORI,G.
Effect of Delivery System on the Pharmacokinetic and Phototherapeutic Properties of Bis(methyloxyethyleneoxy)silicon Phthalocyanine in Tumour-Bearing Mice J.Photochem. Photobiol. B:Biol. 50 (1999) 124-128.

16. RICCHELLI, F., GOBBO, S., MORENO, G. and SALET, C.
Changes of the fluidity of mitochondrial membranes induced by the permeability transition Biochemistry 38: 9295-9300 (1999)

17. BEGHETTO, C., RENKEN, C., ERIKSSON, O., JORI, G., BERNARDI, P. and RICCHELLI, F.
Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies Eur. J. Biochem. 267: 1-9 (2000)

18. MORENO, G., POUSSIN, K., RICCHELLI, F. & SALET, C.
The effects of singlet oxygen produced by photodynamic action on the mitochondrial permeability transition differ in accordance with the localisation of the sensitizer. Arch. Biochem. Biophys. 386: 243-250 (2001)

RICCHELLI, F., CAMERIN, M., BEGHETTO, C. CRISMA, M., MORETTO, V., GOBBO, S., SALVATO, B., SALET, C. & MORENO, G.
Disaccharide modulation of the mitochondrial membrane fluidity changes induced by the membrane potential IUBMB Life 51: 111-116 (2001)

INTERNATIONAL CONFERENCE COMMUNICATIONS

1. RICCHELLI , F., GOBBO, S., JORI,G., SALET,C. & MORENO, G. The partition of hematoporphyrin in mitochondrial membranes: formation of non covalent linear dimers 6th Congress of the European Society for Photobiology , Cambridge ( UK ) 3 - 8 September 1995 , Abs.VI - 1p / 01.

2. MANTAREVA, V., SHOPOVA, M., SPASSOVA, G., WOHRLE, D., MULLER, S., JORI, G., RICCHELLI, F. Pharmacokinetic and photosensitizing properties dependence on the tumor model of cremophor delivered Si(IV) -methoxyethyleneglycol-naphthalocyanine . 12th International Congress on Photobiology, Vienna, Austria , 1-6 -1996, Abs.

3. MORENO, G., SALET, C. and RICCHELLI , F. Effects of Photodynamic Action on Cell Bioenergetics and Calcium Movements 7th Congress of the European Society for Photobiology , Stresa Italy, 8-13 September 1997, Abs. S4 p.13 .

4. BARANYAI, P., KATONA,Z., RICCHELLI, F., JORI, G., BITTER, I. GROFCSIK, A, KUBINYI, M. and VIDOCZY, T. Meso-Tetraarylporphyrins as Molecular Probes of the Microenvironment ibidem, Abs. P107, p. 121.

5. RICCHELLI, F., GOBBO, S., MORENO, G., SALET, C., BRANCALEON, L. and MAZZINI, A. Photophysical Properties of Porphyrin Planar Dimers ibidem, Abs. P136, p.128.

6. RENKEN, C., ERIKSSON, O., RICCHELLI, F. , JORI, G., BERNARDI, P. Photoactivated calcein generates reactive oxygen species (ROS) that are capable of changing Ca2+ accumulation dynamics in isolated rat liver mitochondria. Biophysical Society 42nd annual meeting. Kansas City, Missouri, USA. 22-26 February 1998. Biophysical Journal 74 (2): A382-A382, Part 2

7. RICCHELLI, F., GOBBO, S., MORENO, G., and SALET, C. Changes of membrane fluidity during the mitochondrial permeability transition as probed by hematoporphyrin anisotropy 8th Congress of the European Society for Photobiology, Granada, Spain, 3-8 september 1999 Abs. O77, p.112.

8. BEGHETTO, C., RENKEN, C., ERIKSSON, O., JORI,G., BERNARDI, P. and RICCHELLI, F. Generation of reactive oxygen species by photoactivated calcein. Implications for mitochondrial studies. Photobiologie 2000, Aix-les-Bains, France, 26-27 May 2000 Abs

9. CAMERIN, M., GOBBO, S., SALET, C., MORENO, G: and RICCHELLI, F. Changes of the fluidity of mitochondrial membranes during opening and resealing of the permeability transition pore. Effects of the medium composition. Photobiologie 2000, Aix-les-Bains, France, 26-27 May 2000 Abs

10. BEGHETTO, C., CAMERIN, M., GOBBO, S., TOGNON, G., SALET, C., MORENO, G. and RICCHELLI, F. Reversibility Properties of the Ca2+-induced Permeability Transition in Mitochondria Suspended in Saline and Sugar-Containing Media. IV International Symposium on " Photodynamic Diagnosis and Therapy in Clinical Practice" Bressanone (Italy) 10-13 October, 2001. Abs.P18.

NATIONAL CONFERENCE COMMUNICATIONS

1. RICCHELLI, F. , GOBBO, S., JORI, G. & NIKOLOV, P. Studi spettroscopici sull' interazione di ftalocianine con membrane mitocondriali . Convegno Nazionale Congiunto di Fotobiologia e Fotochimica, Volterra , 6-7 Giugno 1994, Abs. 7S .

2. RICCHELLI, F. GOBBO, S, TOGNON, G. , JORI, G. Spectroscopic studies on the interaction of phthalocyanines and porphyrins with lipid membranes: determination of the nature of dye -binding sites in mitochondrial membranes. XII Congresso SIBPA, Palermo 23 - 28 Settembre 1994 . Abs. D-P10.

3. RICCHELLI,F., GOBBO, S, TOGNON, G., JORI, G., MORENO, G., SALET. Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in mitochondrial membranes. Convegno Annuale della Società Italiana di Fotobiologia , Bressanone (BZ) , 2 - 3 Giugno 1995. Abs. p. 19.

4. RICCHELLI, F., MILANI, M., GOBBO, S., TOGNON, G., JORI, G. , MORENO, G., SALET , C. Effetti della Fotosensibilizzazione con Porfirine sul Trasporto del Ca2+ in Organelli Subcellulari . II Convegno Nazionale Congiunto Fotobiologia e Fotochimica, Maratea (PZ), 2-4 Giugno 1996. Abs. p. 42 .

5. BARBATO, P., GOBBO,S., SALET,C., MORENO,G. and RICCHELLI,F. Porphyrin photodynamic action on Ca2+ transport in endoplasmic reticulum. A comparison with mitochondria. Convegno Annuale della Società Italiana di Fotobiologia, Desenzano (BS), 4-5 Aprile 1998.Abs. p.90.

6. BEGHETTO, C., GOBBO, S., MORENO, G., SALET, C. & RICCHELLI, F. Changes of fluidity of mitochondrial membrane induced by the permeability transition. Congresso Annuale della Società Italiana di Fotobiologia, Pavia, 27-29 Maggio 1999. Abs. p. 29.

7. BEGHETTO, C., CAMERIN, M., GOBBO, S., TOGNON, G., SALET, C., MORENO, G. & RICCHELLI, F. Perturbation of the mitochondrial membrane structure by disaccharides. Congresso Annuale Società Italiana di Fotobiologia, Pisa, 24-26 Maggio 2001. Abs. p. 45

8. CAMERIN, M., POUSSIN, K., GOBBO, S., MORENO, G., RICCHELLI, F. & SALET, C. The microenvironment of the sensitizer in mitochondrial membranes modulates the photodamage induced by singlet oxygen: a study on the permeability transition. Congresso Annuale Società Italiana di Fotobiologia, Pisa, 24-26 Maggio 2001. Abs. p. 45

DOCTORAL DISSERTATIONS

1994

E. Cumerlato Stiffan.: Distribuzione di porfirine e ftalocianine in membrane mitocondriali

1995

M. Milani.: Effetti della fotosensibilizzazione con porfirine sul trasporto del Ca²⁺ in organelli subcellulari

1996

P. Barbato.: Effetti della fotosensibilizzazione con porfirine sul trasporto del Ca²⁺in mitocondri

1997

S. Resoli.:Proprietà fotofisiche di aggregati planari di porfirina

1998

U. Fronzoni.: Effetti della fotosensibilizzazione con porfirine sul trasporto del Ca²⁺ in reticolo endoplasmatico e mitocondri

1999

C. Beghetto.: Variazioni della fluidità delle membrane mitocondriali indotte dalla transizione di permeabilità

2000

1. M. Camerin.: Effetto dell'ossigeno di singoletto sulla transizione di permeabilità mitocondriale: influenza del microintorno del fotosensibilizzatore

2. M. Cescon.: Variazioni indotte dal potenziale di membrana sulla fluidità delle membrane mitocondriali e loro modulazione da parte della composizione del mezzo

TEACHING

1996: Contract professor, University of Teramo, Department of Veterinary Medicine. Course: Optical Spectroscopy in Biomedicine.

INTERNATIONAL PROJECTS

1994-1996. CNR Bilateral Projects. Italy-France (F. Ricchelli- C. Salet): Photodynamic effects on cell physiology.Bioenergetics, calcium transport and cell growth.

1997-1999. CNR Bilateral Projects. Italy-France (F. Ricchelli-G. Moreno): Intracellular photosensitization: photodynamic effects on mitochondria and endoplasmic reticulum.

1998. CNR Bilateral Projects (F. Ricchelli-C. Salet): Photodynamic effects on the calcium transport in cells and subcellular organelles.

1995-1997. Joint research project CNR/Bulgarian Academy of Sciences (F. Ricchelli-M. Shopova): Mechanisms of photodynamic therapy of tumours.

1998. Joint research project CNR/Bulgarian Academy of Sciences (F. Ricchelli-P. Nikolov): Mapping the tryptophan distribution in multitryptophan proteins by steady-state and time-resolved fluorescence spectroscopy.

2001. Joint Research Project CNR/Bulgarian Academy of Sciences (F. Ricchelli/P. Nikolov): Generation of Reactive Oxygen Species by Photoactivated Cell-probes and Implications for Studies of Cellular Functions.

For further information:

Dr. Ricchelli Fernanda
CNR-Institute for Biomedical Technologies
Padova Unit on Metalloproteins
University of Padova
Department of Biology
Via G. Colombo 3,
35121 Padova, Italy

Fax +39 049 8276345
Tel +39 049 8276336

Mail to rchielli@mail.bio.unipd.it