The use of light in medical treatment is nothing new. In the ancient world, roots of the plant Dorstenia were applied topically on to irritated skin which would clear following a few hours of sunshine. The active ingredient, psoralen, is now the basis of PUVA (psoralen + UVA) therapy used to treat the effects of psoriasis and other skin ailments. Psoralen acts by intercalating between base pairs of DNA and upon UV irradiation, the two double bonds form [2+2] cyclo-adducts with thymine, kinking and destroying the DNA of the replicating cells, which was causing the skin irritations in the first place.
In the early twentieth century, following significant progess in synthetic chemistry of coloured dyes throughout the nineteenth century, two German scientists completed work on the toxic effect of eoisin, a methylene based dye. The effect was only noted with the presence of light, and at a later date the presence of oxygen. Thus the modern day science of photodynamic therapy (PDT) was discovered, consisting of three components: photosensitising dye, light, and oxygen. Despite positive results from trials, the work went relatively unnoticed, and it wasn’t until the 1970s that it really picked up again.
The basic principles of PDT are relatively easy to consider. A light absorbing dye is applied or injected into the patient and after a time appropriate for maximum uptake into tumour, the affected area is irradiated with light. The dye absorbs the incident light and an electronically excited state is formed. This subsequently generates a reactive oxygen species (ROS) which destroys the tumour. The concept is beautifully simple, and as the dye is non-toxic in the absence of light, does not carry the negative effect of traditional chemotherapies which are much less discriminate in their action. In PDT, only irradiated areas of body tissue will generate activity leading to cell destruction. As mentioned, there are three components to PDT: light, photosensitiser and oxygen. the latter two are considered in turn, below.
The net result of dye irradiation is the generation of reactive oxygen species, and it is generally considered that singlet oxygen 1O2 is the ROS responsible for cell destruction in PDT. 1O2 is formed when oxygen, which in the ground state exists in as a triplet (3O2 ) absorbs energy. According to the MO diagram of the ground state 3O2 shown, the singlet is formed with the pairing of electrons in the LUMO. This is energetically unstable relative to the ground state, as there is a vacant orbital of the same energy available to the paired electron. Hence, 1O2 is energetically higher (thus more reactive) and will return to its triplet ground state. The lifetime of the singlet state in a cell is of the order of 100s of ns, and it has been estimated that it can diffuse less than 50 nm in this time. Therefore, in cells of the order of microns, the action is limited to cellular dimensions.
1O2 is formed by energy transfer from the photoexcited dye. But an alternative is possible. If the photoexcited dye transfers an electron to molecular oxygen, a superoxide anion is formed. Therefore, a crucial aspect of drug development in PDT is the nature of the ROS formed. Electron transfer forming superoxide anion is called a Type I reaction. Energy transfer forming 1O2 is called a Type II reaction. We can distinguish between these by understanding the chemistry behind them. Importantly for us here. Type II is detected because as singlet oxygen returns to its triplet ground state, it emits a small amount of infrared phosphorescence, which can be detected (see figure – the emission maximum is approximately 1270 nm). Type I on the other hand, can be detected by monitoring the redox chemistry of Fe3+ and subsequent formation of hydroxyl radicals (the photo-Fenton mechanism).
The photosensitiser has several functions. It must locate in the tumour. This involves considering both hydrophilic and lipophilic components in the molecular design that are not covered here. It should also absorb in the far visible region (600 – 800 and preferably 700 – 800 nm). Haemoglobin is a significant component of body tissue, and absorbs strongly in the mid visible region (580 nm). This is obvious when we shine a light through our hand – only red light passes through. Therefore the ideal will be one which absorbs light where the body does not, allowing them to be used deep within body tissue (e.g. liver, pancreas).
As it stands, clinically approved PDT drugs are not yet optimised for longer wavelength light absorption, and hence PDT treatment is currently limited to areas that are easy to expose to a light source: skin, lung, oesophagus, etc. This issue of “penetration depth” was the subject of a recent court case, whereby a doctor justifiably claimed that PDT treatment would not be suitable for treatments such as liver cancer, as the liver was just too big for light to pass through. (The doctor was subsequently acquitted of all charges). The bar chart (Ref 1) shows how photosensitiser absorption capacity affects penetration depth, and this is the focus of current research (below). Penetration doubles once light at longer wavelength than the absorption of haemoglobin is achieved (630 nm) and doubles again at 700 nm. An ideal photosensitiser will therefore absorb between 700 – 800 nm.
Other factors for an ideal photosensitiser include: low toxicity in the absence of light and little post-treatment side affects. One of the most significant side effects is post-treatment light sensitivity, whereby patients have to avoid light for fear that healthy body tissue which have residual amount of photosensitiser present will generate unwanted activity.
From a photochemical point of view, one of the most important dye characteristics is that it will form a high concentration of triplet excited state. Since the mechanism of action is generation of singlet oxygen, a singlet state may not be long-lived enough to allow time for reaction with oxygen (singlet-singlet deactivation via fluorscence or non-radiative means is an allowed process, and therefore very fast). If the triplet forms via intersystem crossing, its deactivation is forbidden, and hence slower, allowing the energy transfer to oxygen to be more competitive. This again provides potential for future research (see below).
The first clinically approved PDT drug was photofrin (R). Photofrin is a porphyrin -based compound and you may wish to examine its structure to identify hydrophilic and lipophilic components alluded to above. It absorbs at 630 nm, which is within the PDT window and has a respectable quantum yield of inter-system crossing of about 25%. However, its absorption is not significant, with a low extinction coefficient. Its worth noting here also that PDT is not limited to cancer based therapy, it has also been used as alternative to antibiotics and for gum disease (There is a good overview article in Chemistry World, ref 2, and some nice pictures for dental treatment at the Periowave site, ref 3)
Photofrin’s limitation is primarily its light absorption. To get to a point where PDT can become more versatile, the photosensitiser needs first to absorb in the 700 – 800 nm window (and then subsequently satisfy all other demands re singlet oxygen generation, accumulation in tumours….). Reduction of one (chlorins) and two (bacteriochlorins) of the four pyrroles in porphyrin based compounds have been found to shift the wavelength of absorption to longer wavelengths. In the example shown, the absorption shifts to about 700 nm for chlorin-based molecules and to about 800 nm for bacteriochlorin based molecules. The compound shown has a high extinction coefficient of absorption and good oxygen generation capabilities. More information on these compounds is available in Ref 1.
Intersystem crossing can be enhanced by the heavy atom effect, and this is the subject of another class of boron-based compounds. It was noted for certain sites of substitution of iodine, the singlet oxygen generation capacity increased, attributed to an increased intersystem crossing yield caused by the iodine heavy atoms. More information on these compounds is available in reference 4.
PDT provides huge potential in treatment of cancerous tumours and a range of other antibiotic treatments. It has been called a very selective surgeon’s knife thanks to its ability to isolate the affected area for treatment with little collateral damage. At the core of future developments of PDT is an understanding of the photochemistry at its heart, and now a century after the first PDT action was discovered, it looks like it has a positive future.
1. (Primary reference for this article) Chem Soc Rev, 2011, 40, 340: Sections A, B, C.1, C.4, D, F.2, F.3
2. Chemistry World, 2012, April, 52, see also Education in Chemistry, 2004, May, 71.
3. Periowave blog (accessed December 2012)
3. Chem Soc Rev, 2013, 42, 77: pages 77 – 81.