Mechanisms

In PDT, the photosensitiser becomes activated by light, but it does not react directly with cells and tissues. Instead, it passes on its energy to molecular oxygen to form a particularly reactive species called 'singlet oxygen'. After it has passed on its energy, the photosensitiser returns to its starting point, where it is available to begin the whole process again. Effectively, therefore, it acts as a kind of catalyst which makes the whole process very efficient. In the design of new drugs and in the optimisation of clinical treatment it is very important to understand precisely what happens to photosensitisers in the body and what's the best drug dose and the best light dose for patient treatment.

PDT combines the preferential accumulation of the photosensitiser in the target tissue with precise illumination, to provide the selectivity of the treatment. The light penetrates the tissue and causes excitation of the photosensitiser.



Properties of light

spectrum of visible light

The spectum of visible light

Activation of the photosensitisers by light is a pre-requisite to successful PDT. The transmission of light through tissue is low at 400 nm because of scattering and absorption by natural chromophores. Light penetration increases with increasing wavelength up to 800 nm. A particular wavelength of light is needed for each photosensitiser to maximise penetration through the tumour and excitation of the photosensitiser.

A small, compact laser

A compact laser light source

PDT light delivery systems have improved in the last 20 years. Tuneable dye lasers are frequently used in investigative studies because they allow maximum flexibility, however, for clinical use they are not ideal due to size and mobility restraints.

Licensing of specific photosensitisers, using one particular wavelength, has led to the development of small compact lasers, such as diode lasers and light emitting diode array lasers which are more convenient to use in clinical situations.

However, superficial lesions can be easily treated with lamps such as the xenon arc lamp. Further development of light applicators compatible with endoscopes and the use of several optical fibres in interstitial therapy of larger tumours, has overcome the penetration difficulties encountered in earlier studies.

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Photosensitiser excitation

Jablonski diagram

Photosensitisers have a stable electronic configuration which is in a singlet state in their lowest or ground state energy level. Following absorption of a photon of light of specific wavelength a molecule is promoted to an excited state, which is also a singlet state and which is short lived. The photosensitiser returns to the ground state by emitting a photon (fluorescence) or by internal conversion with energy loss as heat. It is also possible that the molecule may convert to the triplet state via intersystem crossing which involves a change in the spin of an electron. The triplet state photosensitiser has lower energy than the singlet state, but has a longer lifetime (typically > 500 ns for photosensitisers) and this increases the probability of energy transfer to other molecules.[99]

The tendency of a photosensitiser to reach the triplet state is measured by the triplet state quantum yield, which measures the probability of formation of the triplet state per photon absorbed (depending on the interaction of the singlet species with other substrates producing fluorescent quenching). The triplet state lifetime influences the amount of cytotoxic species produced by collision-induced energy transfer to molecular oxygen and other cellular components. A high intersystem crossing probability will produce an effective population of excited triplet state photosensitiser molecules whose energy can then be transferred by the two mechanisms described below. In addition, the photosensitiser is not destroyed but returns to its ground state without chemical alteration and is able to repeat the process of energy transfer to oxygen many times.

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Type I and II reaction mechanisms

There are two mechanisms by which the triplet state photosensitiser can react with biomolecules; these are known as the Type I and Type II reactions.

Type I involves electron/hydrogen transfer directly from the photosensitiser, producing ions, or electron/hydrogen abstraction from a substrate molecule to form free radicals. These radicals then react rapidly, usually with oxygen, resulting in the production of highly reactive oxygen species (e.g. the superoxide and the peroxide anions). These radicals then attack cellular targets as described below.

Type II reactions produce the electronically excited and highly reactive state of oxygen known as singlet oxygen. Direct interaction of the excited triplet state photosensitiser with molecular oxygen (which, unusually, has a triplet ground state) results in the photosensitiser returning to its singlet ground state and the formation of singlet oxygen.

In PDT, it is difficult to distinguish between the two reaction mechanisms. There is probably a contribution from both Type I and II processes indicating the mechanism of damage is dependent on oxygen tension and photosensitiser concentration.

Mechanisms of PDT


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Cellular photosensitisation

PDT produces cytotoxic effects through photodamage to subcellular organelles and biomolecules. These sites of photodamage may reflect the localisation of the photosensitiser in the cell. A variety of cellular components such as amino acids (particularly cysteine, histidine, tryptophan, tyrosine and methionine), nucleosides (mainly guanine) and unsaturated lipids can react with singlet oxygen. The diffusion distance of singlet oxygen is relatively short (about 0.1 micron), therefore the photosensitiser must associate intimately with the substrate for efficient photosensitisation to occur. Although the Type II process is considered the more relevant reaction mechanism in PDT, cytotoxic species generated by the Type I reaction process can also act in a site-specific manner[100].

Many factors determine the cellular targets of photosensitisers. The incubation parameters and mode of delivery as well as the chemical nature of the photosensitiser can all influence subcellular localisation, creating a number of potential targets for photodamage. In cell culture studies with porphyrin based photosensitisers, short incubation times (up to 1 h) prior to illumination leads primarily to membrane damage whereas extended incubation periods followed by light exposure results in damage to cellular organelles and macromolecules.

Hydrophobic (lipophilic) compounds preferentially bind membranes and will target structures such as the plasma membrane, mitochondria, lysosomes, endoplasmic reticulum and the nucleus. Oxidative degradation of membrane lipids can cause the loss of membrane integrity, resulting in impaired membrane transport mechanisms and increased permeability and rupturing of membranes. Cross-linking of membrane associated polypeptides may result in the inactivation of enzymes, receptors and ion channels.

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Mitochondrial localisation

The mitochondrion has been shown to be a critical target in PDT. Lipophilic porphyrins have demonstrated intimate intracellular association with mitochondrial membranes, whilst cationic compounds such as rhodamines and cyanines may accumulate in these organelles due to mitochondrial membrane potential. Much work has focused on photosensitisation of mitochondria because these organelles perform vital functions in the cell. ATP is synthesised by oxidative phosphorylation in the mitochondria and is required for energy requiring processes such as replication, protein synthesis, DNA synthesis and transport. Mitochondrial photosensitisation may cause the uncoupling of respiration and phosphorylation resulting in the impairment of ATP synthesis and subsequent loss of cellular function. At the molecular level several mitochondrial enzymes and carriers involved in ATP synthesis have displayed sensitivity to mitochondrial photosensitisation. Following PDT the loss of mitochondrial integrity has been observed to occur before the loss of plasma membrane integrity, underlining the importance of the mitochondria as targets for PDT. Mitochondrial damage can also induce nuclear chromatin condensation, and has been linked to the induction of apoptosis.

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Lysosomal localisation

Lysosomal localisation has been observed for a number of photosensitisers. Initially it was thought that cell death was due to the release of enzymes following lysosomal membrane photodamage, however cell survival has since been observed following photodamage to 80% of cellular lysosomes. Recent studies have demonstrated that photosensitisers are redistributed from the lysosomes to other cellular sites upon light exposure[101].

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Nuclear localisation

At the level of the nucleus, PDT has been shown to cause single/double stranded breaks and alkali-labile sites in DNA, as well as induction of sister chromatid exchanges and chromosomal aberrations. However, further studies have indicated that nuclear damage and/or repair is not generally a dominant factor in PDT mediated cytoxicity.

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