Photodynamic therapy
Photodynamic therapy is a form of phototherapy involving light and a photosensitizing chemical substance used in conjunction with molecular oxygen to elicit cell death.
PDT is used in treating acne, wet age-related macular degeneration, psoriasis, and herpes. It is used to treat malignant cancers, including head and neck, lung, bladder and skin.
Advantages lessen the need for delicate surgery and lengthy recuperation and minimal formation of scar tissue and disfigurement. A side effect is the associated photosensitisation of skin tissue.
Basics
PDT applications involve three components: a photosensitizer, a light source and tissue oxygen. The wavelength of the light source needs to be appropriate for exciting the photosensitizer to produce radicals and/or reactive oxygen species. These are free radicals generated through electron abstraction or transfer from a substrate molecule and highly reactive state of oxygen known as singlet oxygen.PDT is a multi-stage process. First a photosensitiser, ideally with negligible toxicity other than its phototoxicity, is administered in the absence of light, either systemically or topically. When a sufficient amount of photosensitiser appears in diseased tissue, the photosensitiser is activated by exposure to light for a specified period. The light dose supplies sufficient energy to stimulate the photosensitiser, but not enough to damage neighbouring healthy tissue. The reactive oxygen kills the target cells.
Reactive oxygen species
In air and tissue, molecular oxygen occurs in a triplet state, whereas almost all other molecules are in a singlet state. Reactions between triplet and singlet molecules are forbidden by quantum mechanics, making oxygen relatively non-reactive at physiological conditions. A photosensitizer is a chemical compound that can be promoted to an excited state upon absorption of light and undergo intersystem crossing with oxygen to produce singlet oxygen. This species is highly cytotoxic, rapidly attacking any organic compounds it encounters. It is rapidly eliminated from cells, in an average of 3 μs.Photochemical processes
When a photosensitiser is in its excited state it can interact with molecular triplet oxygen and produce radicals and reactive oxygen species, crucial to the Type II mechanism. These species include singlet oxygen, hydroxyl radicals and superoxide ions. They can interact with cellular components including unsaturated lipids, amino acid residues and nucleic acids. If sufficient oxidative damage ensues, this will result in target-cell death.Photochemical mechanisms
When a chromophore molecule, such as a cyclic tetrapyrrolic molecule, absorbs a photon, one of its electrons is promoted into a higher-energy orbital, elevating the chromophore from the ground state into a short-lived, electronically excited state composed of vibrational sub-levels. The excited chromophore can lose energy by rapidly decaying through these sub-levels via internal conversion to populate the first excited singlet state, before quickly relaxing back to the ground state.The decay from the excited singlet state to the ground state is via fluorescence. Singlet state lifetimes of excited fluorophores are very short since transitions between the same spin states conserve the spin multiplicity of the electron and, according to the Spin Selection Rules, are therefore considered "allowed" transitions. Alternatively, an excited singlet state electron can undergo spin inversion and populate the lower-energy first excited triplet state via intersystem crossing ; a spin-forbidden process, since the spin of the electron is no longer conserved. The excited electron can then undergo a second spin-forbidden inversion and depopulate the excited triplet state by decaying to the ground state via phosphorescence. Owing to the spin-forbidden triplet to singlet transition, the lifetime of phosphorescence is considerably longer than that of fluorescence.
Photosensitisers and photochemistry
Tetrapyrrolic photosensitisers in the excited singlet state are relatively efficient at intersystem crossing and can consequently have a high triplet-state quantum yield. The longer lifetime of this species is sufficient to allow the excited triplet state photosensitiser to interact with surrounding bio-molecules, including cell membrane constituents.Photochemical reactions
Excited triplet-state photosensitisers can react via Type-I and Type-II processes. Type-I processes can involve the excited singlet or triplet photosensitiser, however due to the short lifetime of the excited singlet state, the photosensitiser can only react if it is intimately associated with a substrate. In both cases the interaction is with readily oxidisable or reducible substrates. Type-II processes involve the direct interaction of the excited triplet photosensitiser with molecular oxygen.Type-I processes
Type-I processes can be divided into Type I and Type I. Type I involves the transfer of an electron from a substrate molecule to the excited state photosensitiser, generating a photosensitiser radical anion and a substrate radical cation. The majority of the radicals produced from Type-I reactions react instantaneously with molecular oxygen, generating a mixture of oxygen intermediates. For example, the photosensitiser radical anion can react instantaneously with molecular oxygen to generate a superoxide radical anion, which can go on to produce the highly reactive hydroxyl radical, initiating a cascade of cytotoxic free radicals; this process is common in the oxidative damage of fatty acids and other lipids.The Type-I process involves the transfer of a hydrogen atom to the excited state photosensitiser. This generates free radicals capable of rapidly reacting with molecular oxygen and creating a complex mixture of reactive oxygen intermediates, including reactive peroxides.
Type-II processes
Type-II processes involve the direct interaction of the excited triplet state photosensitiser with ground state molecular oxygen ; a spin allowed transition—the excited state photosensitiser and ground state molecular oxygen are of the same spin state.When the excited photosensitiser collides with molecular oxygen, a process of triplet-triplet annihilation takes place. This inverts the spin of one oxygen molecule's outermost antibonding electrons, generating two forms of singlet oxygen, while simultaneously depopulating the photosensitiser's excited triplet state. The higher-energy singlet oxygen state is very short-lived and rapidly relaxes to the lower-energy excited state. It is, therefore, this lower-energy form of singlet oxygen that is implicated in cell injury and cell death.
The highly-reactive singlet oxygen species produced via the Type-II process act near to their site generation and within a radius of approximately 20 nm, with a typical lifetime of approximately 40 nanoseconds in biological systems.
It is possible that singlet oxygen can diffuse up to approximately 300 nm in vivo. Singlet oxygen can theoretically only interact with proximal molecules and structures within this radius. ROS initiate reactions with many biomolecules, including amino acid residues in proteins, such as tryptophan; unsaturated lipids like cholesterol and nucleic acid bases, particularly guanosine and guanine derivatives, with the latter base more susceptible to ROS. These interactions cause damage and potential destruction to cellular membranes and enzyme deactivation, culminating in cell death.
It is probable that in the presence of molecular oxygen and as a direct result of the photoirradiation of the photosensitiser molecule, both Type-I and II pathways play a pivotal role in disrupting cellular mechanisms and cellular structure. Nevertheless, considerable evidence suggests that the Type-II photo-oxygenation process predominates in the induction of cell damage, a consequence of the interaction between the irradiated photosensitiser and molecular oxygen. Cells in vivo may be partially protected against the effects of photodynamic therapy by the presence of singlet oxygen scavengers. Certain skin cells are somewhat resistant to PDT in the absence of molecular oxygen; further supporting the proposal that the Type-II process is at the heart of photoinitiated cell death.
The efficiency of Type-II processes is dependent upon the triplet state lifetime τT and the triplet quantum yield of the photosensitiser. Both of these parameters have been implicated in phototherapeutic effectiveness; further supporting the distinction between Type-I and Type-II mechanisms. However, the success of a photosensitiser is not exclusively dependent upon a Type-II process. Multiple photosensitisers display excited triplet lifetimes that are too short to permit a Type-II process to occur. For example, the copper metallated octaethylbenzochlorin photosensitiser has a triplet state lifetime of less than 20 nanoseconds and is still deemed to be an efficient photodynamic agent.
Photosensitizers
Many photosensitizers for PDT exist. They divide into porphyrins, chlorins and dyes. Examples include aminolevulinic acid, Silicon Phthalocyanine Pc 4, m-tetrahydroxyphenylchlorin and mono-L-aspartyl chlorin e6.Photosensitizers commercially available for clinical use include Allumera, Photofrin, Visudyne, Levulan, Foscan, Metvix, Hexvix, Cysview and Laserphyrin, with others in development, e.g. Antrin, Photochlor, Photosens, Photrex, Lumacan, Cevira, Visonac, BF-200 ALA, Amphinex and Azadipyrromethenes.
The major difference between photosensitizers is the parts of the cell that they target. Unlike in radiation therapy, where damage is done by targeting cell DNA, most photosensitizers target other cell structures. For example, mTHPC localizes in the nuclear envelope. In contrast, ALA localizes in the mitochondria and methylene blue in the lysosomes.