Effect of aggregation state on photophysical and photochemical properties of sensitizers.

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The 5,10,15,20-meta-tetra(hydroxyphenyl)chlorin

mTHPC is a second-generation photosensitizer (Bonnett et al. 1989) and is one of the most effective sensitizers studied to date (Dougherty et al. 1998). It mediates cell photodamage, principally through singlet oxygen formation (Melnikova, Bezdetnaya et al. 1999) and its efficacy is sensitive to oxygenation conditions (Coutier et al. 2002). mTHPC has been granted European approval for palliative treatment of patients with advanced head and neck cancers and undergoes clinical open-label multicenter studies for the treatment of early squamous cell carcinoma (Copper et al. 2003; Hopper et al. 2004).
mTHPC is introduced to patients intravenously. mTHPC’s hydrophobic nature defines its affinity to plasma proteins. Hence, the interactions with plasma components and blood cells can play an important role in mTHPC-PDT efficacy. Studies on mTHPC interaction with plasma protein fractions are sparse (Michael-Titus et al. 1995; Hopkinson et al. 1999; Kessel 1999). mTHPC displays some unusual properties in vitro and in vivo compared with many other sensitizers. Gradient-density ultracentrifugation demonstrated the presence of weakly fluorescing aggregated mTHPC species in the regions of albumin or HDL/albumin (Hopkinson et al. 1999; Kessel and E. Sykes 1999). mTHPC forms large-scale aggregates in aqueous media that monomerize upon interaction with plasma proteins (Bonnett et al. 2001). This sensitizer is rigidly fixed in model membranes and strongly retained in cells in vitro (Ball et al. 1999; Bombelli et al. 2005). mTHPC displays an unusual pharmacokinetic behaviour in human and rabbit plasma with a secondary peak at about 10 and 6 h after in intravenous injection, respectively (Ronn et al. 1997; Glanzmann et al. 1998). These phenomena were supposed to be explained by initial retention of PS in the liver or sensitizer aggregates in the vasculature. Similar pharmacokinetic profile was reported only for hexyl-ether derivative of pyropheophorbide-a in mice (Bellnier et al. 1993). MTHPC has small initial volume of distribution with high retention in the vasculature together with two peaks of PDT efficacy (2h and 24h) in mice (Jones et al. 2003).
It has been demonstrated that the Golgi apparatus and endoplasmic reticulum (ER) are preferential sites of mTHPC accumulation in MCF-7 human adenocarcinoma cells after 3h of incubation (Teiten, Bezdetnaya et al. 2003). Golgi apparatus and ER were shown to be the primary PDT-induced damage sites as measured by enzymes photoinactivation technique (Teiten, Bezdetnaya et al. 2003; Teiten, Marchal et al. 2003). Damage to Golgi apparatus was confirmed by fluence-dependent alterations of Golgi apparatus and mitochondria morphology (Melnikova, Bezdetnaya, Bour et al. 1999). Both apoptotic and necrotic pathway are implicated in mTHPC-mediated HT29 cell photoinactivation that is governed by mitochondrial membrane photodamage manifested by cytochrome C release and dissipation of mitochondrial membrane potential (Marchal et al. 2005).
During irradiation at 650 nm the absorption spectra of mTHPC in organic, PBS and PBS containing 10% FCS the major absorption bands at 380-450 and 650 nm decreased (Hadjur et al. 1998). A new absorption band was observed at 320 nm, attributed to the formation of a photoproduct. The spectra of mTHPC fluorescence also decreased upon irradiation but no fluorescent photoproducts were detected. A strong dependence of the photodegradation on oxygen concentration and the formation of photoproducts have been reported (Hadjur et al. 1998). Hadjur et al. determined the quantum yields of photobleaching ФPb in aqueous solution containing 10 % FCS to be 1.54 x 10-5 for air saturated conditions and 1.8 x 10-6 after N2 bubling. In aerobic conditions the photodegradation, as well as the formation of photoproducts, have been competitively inhibited by singlet oxygen quenchers. On the basis of photobleaching experiments Handjur et. al. also determined the quantum yield of singlet oxygen production (ФΔ) by mTHPC, which appeared to be 0.3 in ethanol and 0.01 in PBS suggesting that mTHPC is highly aggregated in aqueous media (Hadjur et al. 1998). Products of mTHPC oxidation irradiated in methanol have been separated and identified by high-performance liquid chromatography (HPLC). The major compound of oxygenation process has been described as β-hydroxy-mTHPC with an absorption band around 423 nm (Jones et al. 1996). MTHPC has been reported to be a moderately photolabile compound. A comparative study of mTHPBC and mTHPC in methanol–water (3:2, v/v) solution demonstrated a 90 fold greater mTHPBC photobleaching rate compared to mTHPC (Bonnett, Djelal et al. 1999). Rovers et al. in an in vivo study on Colo 26 tumour bearing mice showed that the rate of bleaching of mTHPBC was approximately 20 times greater than that of mTHPC (Rovers, de Jode, Rezzoug et al. 2000). The ФPb value for mTHPC in PBS with 10 % FCS solution is an order of magnitude lower compared to BPD-MA (ФPb = 2.07 x 10-4) (Aveline et al. 1994).
mTHPC has a strong absorbance in the red region (650 nm) with high molar extinction coefficient (Table 2.2) (Bonnett, Djelal et al. 1999). This offers promising therapeutic perspectives for PDT of deep tumours and pigmented tissues. Pre-clinical studies have demonstrated that in female BALB/c mice bearing PC6 tumour cells the depth of necrosis was 3.79 ± 0.28 mm for mTHPC dose of administered photosensitizer 0.375 µmol/kg for mTHPC (Bonnett et al. 1989). Another in vivo study demonstrated that area of necrosis after irradiation of mTHPC-sensitised liver is 26 ± 4 mm2 (Rovers, de Jode and Grahn 2000). The absence of correlation between PS concentration in tumor and PDT efficiency was observed in vivo (Veenhuizen et al. 1997; Ris et al. 1998).

Cells and tissue damage effects of PDT

PDT induces both direct and indirect antitumor effects (Castano et al. 2005). It can directly destroy tumor cells that undergo apoptosis and necrosis accompanied by induction of the inflammatory response and a slowly developing adaptive immunity that can potentiate local antitumor effects and might possibly induce systemic immunity. PDT together with inflammatory response can also damage tumor vasculature leading to the early vascular shutdown and ischemia-related cell death.

Vascular Shutdown and Inflammation

The alteration of endothelial cells during PDT treatment seems to be the origin of modifications observed in vasculature (Fingar et al. 2000). PDT provokes modifications of organisation of the proteins of cytoskeleton of endothelial human cells with consecutive induction of calcium influx in cells (Foster et al. 1991). The modifications of cytoskeletal proteins induce the changes of cells form and the loss of intracellular communications (Fingar et al. 2000). Such changes serve as a signal to the platelets and neutrophils activation which adhere on the vessel wall, roll toward the constriction and aggregate, at which point they migrate into the surrounding tissues following chemokine gradients (Steele et al. 1985). After adhesion platelets release a great quantity of vasoactive molecules such as thromboxan which amplify platelets aggregation being powerful vasoconstrictor (Fingar et al. 1992). In the region of injury cascades of eicosinoids lead to vessel constrictions. The formation of space between endothelial cells contribute to the reduction of tumoral perfusion and vascular permeability (McMahon et al. 1994; Zilberstein et al. 2001). It was demonstrated that vascular destruction occurred to a greater extent in vivo (Henderson et al. 1984). This cause the blood stasis and tumor cells starvation of oxygen and nutrients and reduce the survivability of cells in vivo (Henderson et al. 1985; Henderson and Fingar 1987; Fingar et al. 1992). It was reported that vascular destruction after PDT is accompanied by inflammatory response like after tissue injury (Korbelik 1996).
Different photosensitizers do not produce the same type of vascular response: NPe6-PDT produce blood stasis mainly due to platelets aggregated on the artery walls while SnEt2 produces an inflammatory response without vessel constriction or platelet aggregation (McMahon et al. 1994). Vascular destruction is generally considered to be one of the major effects contributing to tumor destruction.

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Direct cell destruction

One of the first who provided the evidence that cells may undergo two distinct types of cell death was Kerr (Kerr et al. 1972). The first type is known as necrosis, a violent and quick form of death affecting extensive cell populations, characterized by cytoplasm swelling, destruction of organelles and disruption of the plasma membrane, leading to the release of intracellular contents and inflammation. Necrosis has been referred to as accidental cell death, caused by physical or chemical damage and has generally been considered an unprogrammed process. During necrosis decomposition of cell is principally mediated by proteolytic activity (Castano et al. 2005).
Several types of cell death were termed apoptosis or programmed cell death (Agostinis et al. 2004; Almeida et al. 2004). They are identified in single cells usually surrounded by healthy-looking neighbors, and characterized by cell shrinkage, blebbing of the plasma membrane, the organelles and plasma membrane retain their integrity for quite a long period. As a rule the apoptotic program initiated by PDT is the rapid release of mitochondrial cytochrome C into the cytosol followed by activation of the apoptosome and procaspase 3. In vitro, apoptotic cells are ultimately fragmented into multiple membrane-enclosed spherical vesicles. In vivo, these apoptotic bodies are scavenged by phagocytes, inflammation is prevented. Apoptosis, requires transcriptional activation of specific genes, include the activation of endonucleases, consequent DNA degradation into oligonucleosomal fragments, and activation of caspases. Some alternative modes of cell death have bee described: mitotic cell death (Castedo et al. 2004), programmed necrosis (Bizik et al. 2004), cathepsin-mediated lysosomal death pathway (Leist and Jaattela 2001) and autophagic cell death (Yu et al. 2004).
Photosensitizers that localize in cellular organelles such as endoplasmic reticulum or mitochondry can induce apoptosis via photodamage of Bcl-2 and Bcl-xl proteins (Kessel and Luo 1999). With PS localized in the plasma membrane, the photosensitization process can be switched to the necrotic cell death likely due to loss of plasma membrane integrity and rapid depletion of intracellular ATP (Kessel and Poretz 2000; Agostinis et al. 2004). It is believed that lower dose PDT leads to more apoptosis, while higher doses provoke more necrosis (Plaetzer et al. 2002). Cells sufficiently damaged by PDT are killed, regardless of the mechanism involved. This means that inhibition of apoptosis reorients cells to necrotic pathway, but cannot increase cell survival (Thibaut et al. 2002).

Photophysical and photochemical properties of sensitizers

Photobleaching of sensitizers

During the photodynamic treatment in addition to the reaction of PS with biological substrate, self-photosensitization occurs, the reactive oxygen intermediates can interact with the photosensitizer, leading to its transformation and/or destruction. This phenomenon is called photobleaching. Photobleaching is relevant to a variety of fields, from laser technology to photomedicine. The first observation of photobleaching in the photodynamic therapy field was made in 1986 by Moan (Moan 1986). Photobleaching is usually observed as lowering of the optical density or the fluorescence intensity of the solution during irradiation (Spikes 1992; Rotomskis et al. 1996). Two types of photobleaching can be considered (Bonnett and Martínez 2001):
– photomodification, where loss of absorbance or fluorescence during irradiation leads only to PS transformation into modified form.
– “true photobleaching”, where chemical change is profound and results in small fragments that do not absorb in the visible spectral region.

Table of contents :

I GENERAL INTRODUCTION
II INTRODUCTION
II.1. HISTORY AND CLINICAL APPLICATIONS OF PHOTODYNAMIC THERAPY
II.2. PHOTOSENSITIZATION MECHANISMS
2. 1. Pathways of molecular excitation and deactivation.
2. 2. Mechanisms of photosensitized reactions.
2.2.1. Types I photosensitization mechanism.
2.2.2. Types II photosensitization mechanism.
2. 3. The properties of an ideal photosensitizer.
2. 4. Tetraphenylchlorin series photosensitizers.
2. 4. 1. The 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin.
2. 5. Cells and tissue damage effects of PDT.
2. 5. 1. Vascular shutdown and inflammation.
2. 5. 2. Direct cell destruction.
II.3. PHOTOPHYSICAL AND PHOTOCHEMICAL PROPERTIES OF SENSITIZERS .24
3. 1. Photobleaching of sensitizers.
3. 1. 1. Parameters effecting photobleaching: aggregation state, pH, ionic strength and oxygen concentration.
3. 2. Effect of aggregation state on photophysical and photochemical properties of sensitizers.
3. 3. Photophysical properties of porphyrinoid sensitizer non-covalently bound to proteins.
3. 4. Electronic structure of porphyrinoid photosensitizers.
II.4. PHOTOSENSITIZERS INTERACTIONS WITH PLASMA PROTEINS
4. 1. Pharmacokinetics of sensitizers.
4. 2. Kinetic and equilibrium characteristics of sensitizers interactions with proteins.
4. 2. 1. Mechanisms of sensitizers redistribution between plasma proteins .
4. 2. 1. 1. Collision mechanism.
4. 2. 1. 2. Redistribution through the aqueous phase
4. 2. 2. Thermodynamics of sensitizers redistribution between plasma proteins. Eyring theory.
II.5. INTRACELLULAR LOCALISATION OF PHOTOSENSITIZERS
5. 1. Techniques to study sensitizer intracellular localisation and aggregation state
5. 1. 1. Confocal laser scanning fluorescence microscopy.
5. 1. 2. Fluorescence lifetime imaging microscopy (FLIM).
5. 2. Sub-cellular localisation and dynamics of sensitizers during PDT.
5. 2. 1. Sites of sub-cellular localization of hydrophilic and hydrophobic sensitizers.
5. 2. 2. Relocalisation of sensitizers upon irradiation.
III OBJECTIVES
IV RESULTS
IV. 1. MTHPC-based photodynamic treatment in vivo.
IV. 2. Investigation of mTHPC interactions with plasma proteins.
IV. 3. Redistribution of mTHPC from plasma proteins to model membranes
IV.4. Calculation of quantum yield of MCF-7 cells inactivation by mTHPC-PDT: influence of incubation time and sensitizers localization.
IV.5. Theoretical and experimental study of the effects of solvent on the electronic structure of tetrapyrrole compounds: application for the determination of the structure of aggregates.
V GENERAL DISCUSSION.
VI CONCLUSION AND PERSPECTIVES

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