Erythrocytes—the ‘house elves’ of photodynamic therapyPhotodynamic therapy (PDT) and fluorescence diagnosis (FD) are being developed for a number of clinical applications. Since fluorophores and photosensitising drugs are usually given systemically their effect on blood elements are of significant importance.Photodynamic effects on erythrocytes occur naturally in patients with erythropoietic protoporphyria (EPP). Exposure to small fluences, as obtained by the erythrocytes when they pass capillaries in the skin, leads to transfer of the photosensitiser protoporphyrin IX (PP IX), from EPP erythrocytes to endothelial cells. Thus, the erythrocytes are partly protected while the endothelial cells suffer photodamage.During photodynamic therapy in vivo erythrocytes are regularly photosensitised. This side effect is partly intended but mostly unwanted, and a summary of this topic is given. Furthermore, the effect of UV-A on erythrocytes that is accompanied with the formation of bilirubin is reviewed. Erythrocytes serve as convenient model cells for experimental research. Such use of erythrocytes to screen new photosensitisers may be of limited value. A combination of photohaemolysis and haemoglobin oxygenation may become the basis for an assay for in vitro phototoxicity.Erythrocytes from birds are good model cells for exploration of physiological and molecular mechanisms involved in PDT. A potential mechanism of PDT induced behaviour resembling apoptosis in erythrocytes is provided.PDT for sterilisation of erythrocyte concentrates has a potential for medical use. Photodynamic effects on the erythrocytes themselves should be avoided. This is realised by choosing a virus-selective photosensitiser, low fluences and treatment of the concentrates with agents like dipyridamole and antioxidants.Future aspects of applications of photosensitisation of red blood cells are discussed.Photodynamic effects are defined as processes initiated by absorption of light in a dye molecule, a photosensitiser, followed by transfer of excitation energy from the dye molecule to oxygen, whereby the strongly oxidising and short lived agent singlet oxygen (1O2) is formed[1,2].1O2 can react with and destroy a number of biomolecules,[3,4] including lipids, proteins and DNA.
Photodynamic effects in living cells are probably almost as old as the origin of life. The first scientific description of the effect was published by von Tappeiner and Raab in .1900[5,6] Although the therapeutic potential of the photodynamic effect was recognised almost from the beginning,[7] it took nearly a whole century of research until the first photosensitiser received marked approval for photodynamic therapy (PDT) of cancer in December .1995[8] In 1913 Meyer-Betz used intravenous injection of haematoporphyrin to demonstrate in his self-experiment the connection between diseases related to haem metabolism and photosensitisation of skin.[9]
As first described in 1953, an error in the last step of haem synthesis leads to fluorescent erythrocytes.[10,11] This inherited disease is called erythropoietic protoporphyria (EPP) according to the definition of Magnus et al. in .1961[12] However, the first documentation of EPP dates back to .1926[13,14] Since photodynamic effects in erythrocytes are characteristic for EPP, a section of this paper is dedicated to this disease.
Potential possibilities for the use of the photosensitation in therapy were explored in the 1940s. This research was revived in the mid-seventies, partly due to the advent of lasers and fibre optic systems.[15] Since that time numerous photosensitisers have been studied and tested for both PDT and fluorescence diagnosis (FD). Red blood cells, or blood in general, are often involved in PDT: (i) Blood is often used to transport the photosensitiser to the tissue.[16] (ii) It is believed that the generation of reactive oxygen species, notably singlet oxygen, is the main molecular mechanism of photodynamic therapy.[17] Therefore, oxygen is needed for efficient PDT. Red blood cells are of particular interest since they constitute the oxygen transport system of the body. Differences in the spectra of oxygenated and deoxygenated haemoglobin can be used to monitor the oxygenation of the tissue under treatment. (iii) The blood is used to control the sensitiser distribution. After topical application of dyes the penetration depth of the dyes can be estimated from fluorescence spectra and from measurement of the speed by which the dyes reach the blood. Liquid chromatography is a convenient method to determine photosensitiser concentrations in blood.[18] (iv) In photodynamic treatment of systemically applied photosensitisers, photodynamic damage to erythrocytes may occur as a side-effect of PDT. This topic is summarised in the section ‘Photodynamic damage to erythrocytes as a side effect of in vivo photodynamic therapy’. However, none of the three first mentioned relations cause considerable photodynamic effects in red blood cells and are therefore not further discussed in this paper.
Upon irradiation with UV-A erythrocytes turn fluorescent. This is most probably due to the formation of bilirubin. The role of bilirubin in PDT is discussed in a separate paragraph.
Red blood cells often serve as model cells for in vitro PDT studies. A recent investigation showed that even though erythrocytes are not ideal cells to evaluate or quantitatively compare photosensitisers, they are useful to elucidate the cellular and molecular principles of PDT.[19] This topic is discussed in the section ‘Red blood cells as model cells in experimental PDT research’.
In the mid-1980s PDT was introduced in the field of virus destruction.[20] Soon afterwards the potential for decontamination of blood components and blood bank-applications was recognised and models for viral inactivation in blood were developed.[21–23] The need for bacterial decontamination of blood components is rising. Similar as for EPP it is advantageous to avoid photodynamic effects in erythrocytes. Decontamination of red blood cell concentrates is reviewed in the section ‘Sterilisation of red blood cell concentrates’.
Finally ideas for further research on photodynamic effects in red blood cells are given.
Erythropoietic protoporphyriaThe porphyrias are metabolic diseases caused by disorders in the biosynthesis of haem.[24] Interestingly, haem, a ferrous protoporphyrin constituting the prosthetic group of haemoglobin, is synthesised in all mammalian cells, with the exception of mature red blood cells, which do not contain mitochondria.[25] The haem synthesis pathway is well known[26] and is taken advantage of in PDT with 5-aminolaevulinic acid (ALA). ALA is a naturally occurring early metabolite in the pathway. When applied exogenously, ALA induces the formation of protoporphyrin IX (PP IX).[27]
Impaired activity of the enzyme ferrochelatase, which inserts the iron into PP IX, causes EPP. The ferrochelatase activity of EPP patients is decreased by 70 to 90% compared to that of controls.[28–30]
The main part of haem synthesis takes place in the bone marrow, and haem accumulates in the maturing red blood cells during the phase of active haemoglobin synthesis.[31] When the red blood cells enter the circulation, PP IX diffuses across the cell membrane and binds to the plasma proteins albumin and haemopexin.[32] Although the intracellular PP IX concentration in erythrocytes decreases with cell age,[33] photosensitisation may take place under light exposure. This effect is illustrated in Fig. 1. It does not lead to haemolysis of erythrocytes but to a light-triggered displacement of protoporphyrin from erythrocytes to endothelial cells. In a series of publications Brun, Sandberg and colleagues elucidated the process by in vitro experiments and proposed the following mechanisms:[34–39] PP IX in erythrocytes is haemoglobin-bound, rather than membrane bound. When exposed to light, the binding site of PP IX is destroyed. Since erythrocytes in vivo receive light while circulating in the upper dermal capillaries, they are exposed to pulses rather than to continuous irradiation. Therefore the (haemoglobin-) released PP IX can diffuse through the cell membrane while the erythrocytes move through the ‘dark’ part of the circulation. Thus, they get no membrane damage. PP IX is then bound to plasma proteins, like albumin and haemopexin, and further delivered to endothelial cells. Additionally, PP IX can be directly transferred from the erythrocyte membranes to the membranes of endothelial cells: Endothelial cells, as well as erythrocytes, have smooth cell surfaces without microvilli. Since the erythrocytes, normally having a diameter of 7 μm, are deformed during their passage through capillaries with a diameter of 4–5 μm, a close contact between the erythrocyte membrane and the endothelial cell membrane is established that facilitates transfer.
These mechanisms result in the described clinical symptom: some EPP patients were able to tolerate several hours of sun exposure one day, while the next day only a few minutes exposure were enough to produce photodynamic effects in the sun-exposed skin.[40]
As a treatment of the photosensitivity sun protection agents, like dihydroxyacetone, as well as quenchers of reactive oxygen species, like beta-carotene, have been applied, both with limited success[41–43].
Photodynamic damage to erythrocytes as a side effect of in vivo photodynamic therapyDamage to erythrocytes was reported after PDT with Photofrin II of cat brain in .1987[44] The erythrocytes appeared in tightly packed aggregates, which many times filled the luminal space completely. Individual erythrocytes of the aggregates occasionally showed morphological evidence of intravascular haemolysis. In 1990 Orenstein et al[45]. found that the lumen of the vessels were filled with swollen erythrocytes after PDT of chicken comb.
These examples show that erythrocytes may be part of a mechanism inducing necrosis in tumours by blocking the vasculature.[46–48] Erythrocyte aggregation might be triggered by a mechanism discussed in the section ‘Red blood cells as model cells in experimental PDT research’ or by photodamage to endothelial cells leading to release of clotting factors.[49] The latter mechanism is similar to the first mentioned one.[50]
The problem of unwanted photodamage to erythrocytes after systemic application of a photosensitiser could be avoided by using highly tissue specific photosensitisers or by application of third generation photosensitisers. These are derivatives of second generation photosensitisers introduced into or attached to chemical devices. This modification increases the biological specificity to deliver photosensitisers to a defined cell type. Therefore, photodamage of the erythrocyte during the transit of the photosensitiser can be avoided or at least significantly reduced.
The UV-A induced formation of bilirubin and its photodegradationA photodynamic effect on erythrocytes occurs after UV-A irradiation in vitro: Erythrocytes, when irradiated with UV-A, turn fluorescent. A fluorescence imaging experiment showing this effect is provided in Fig. 2, parts A and B. Microscopic spectroscopy shows that the fluorescence is emitted at wavelengths between 440 and 740 nm with a maximum at 508 nm (Fig. 2, part C). Due to the biochemical decomposition of haemoglobin[51] and the shape of the spectrum[52] it seems obvious that the UV-A induced fluorescence in erythrocytes is caused by bilirubin type photoproducts of haemoglobin. There are two well described photodynamic effects of bilirubin:
(i) The photosensitising effect of bilirubin is described for in vitro systems. It is an efficient photosensitiser in general[53,54] and especially in erythrocytes.[55] UV-A penetrates the skin down to the dermis.[56] Therefore, blood vessels, and hence erythrocytes, can receive UV-A irradiation when the skin is irradiated, for example in UV-A treated psoriasis-patients or during extensive sun-bathing. However, the clinical reports of erythrocyte damage after UV-A irradiation are missing. This is most probably because of the short time window of the dermal passage of the erythrocytes that does not allow the generation of bilirubin and its photodynamic action. The situation seems to be different when bilirubin is already present in the blood (see below).
(ii) In vivo photodegradation of bilirubin (not exclusively by UV-A, more prominent by blue light) is observed in jaundiced newborns. The phototherapy concept for newborns suffering from hyperbilirubinaemia[57] is in accordance with Fig. 2 (parts A and B) showing a fast bilirubin photodegradation or conversion in non-fluorescent isoforms.
Although the bilirubin in the newborns originates from erythrocytes,[57] the photodynamic effect on erythrocytes is limited. However, it was shown that bilirubin is bound by human erythrocytes.[58] There are clinical reports of photodynamic damage on erythrocytes after phototherapy.[59,60] Such damage, namely the osmotic fragility, was confirmed in an animal model[61] as well as in vitro.[62]
Bruzell[63] recently reviewed the role of bilirubin in jaundice.
Red blood cells as model cells in experimental PDT researchErythrocytes are popular model cells since they are easy to obtain in large numbers with minimal consequences for the donor. They are the most easily available living cells from humans.[64] In connection with the action of photosensitisers they can be used as model cells, either intact or as erythrocyte ghosts (i.e. with the haemoglobin washed out, so that only the membrane and the cytoskeleton remain). Examples are given in Fig. 3 and 4.
Fig. 3 shows data for several tetraphenylporphyrins, which were studied with respect to their ability to accumulate in biological membranes retaining their photophysical properties. Fluorescence lifetimes in erythrocyte ghosts were measured and compared with the corresponding lifetimes in organic solvents. Part A shows the structural formula of unsubstituted tetraphenylporphyrin, a totally ‘flat’ molecule. In contrast, diethyltetraphenylporphyrin and tetraethyltetraphenylporphyrin (Fig. 3B) have a pronounced non-planar, three-dimensional structure. The data are derived from X-ray scattering experiments. However, there were no differences in the magnitude of the fluorescence lifetimes (Fig. 3C) in the solvents compared to the ghosts. The differences in fluorescence lifetimes among different dyes are due to a different bending of the dye molecules.[65] The long fluorescence lifetimes of dyes incubated in erythrocyte ghosts show that mainly monomers accumulate in the erythrocyte membranes.[66] Another application of erythrocyte ghosts as model membranes is related to the generation of singlet oxygen yield during photodynamic action. The generation of singlet oxygen during PDT is known at least since the 1960s.[67,68] Later it was also shown to occur in red blood cells.[69] Oelckers et al. were able to detect singlet oxygen luminescence from the inside of a membrane in erythrocyte ghosts.[70] This was the first demonstration ever of singlet oxygen luminescence originating from inside a native membrane.
Fig. 4 shows structurally similar tetrapyrroles, zinc tetrabenzoporphine and zinc phthalocyanine (part A), confocal sections of erythrocytes loaded with the two dyes (part B) and the erythrocyte survival curves after incubation with zinc tetrabenzoporphine and zinc phthalocyanine and exposure to red light (part C). Benzoporphyrin derivatives as well as zinc phthalocyanines are effective photosensitizers in cell cultures.[71,72] In erythrocytes, zinc phthalocyanine is more efficient than zinc tetrabenzoporphine since the cell survival is reduced to 0.001 after 60 and 120 min irradiation, respectively.[73] Sometimes it is informative to combine erythrocyte ghost experiments and measurements on intact erythrocytes:[74] To measure the accumulation of a dye (e.g. PP IX) in the membrane in intact red blood cells is difficult because of the presence of haemoglobin. However, such measurements are easy to carry out in erythrocyte ghosts. Accumulation of the photosensitiser during incubation can be followed in erythrocyte ghosts and cell survival can be monitored by measuring haemolysis (Fig. 4C). However, this practice is controversial.[75]
Photodynamic damage to erythrocyte membranes starts with potassium release. Haemolysis just indicates disruption of the cell membrane. Ball et al[76]. found that potassium release and haemoglobin release are just time shifted with respect to each other. This indicates that these processes are causally connected. In accordance with the common practice in cell-culture assays, where cell death is monitored by membrane disruption (staining and counting of either dead or living cells),[77] haemolysis of red blood cells can be regarded as a mode of measuring death of red blood cells. Spectroscopic measurements of haemoglobin are much easier and faster to carry out than cell staining and counting. This is another advantage of using red blood cells for this type of investigations.
The mechanisms behind the above mentioned release of potassium prior to erythrocyte haemolysis are unknown. However, we propose a mechanism related to an operation of a non-selective cation channel[78] in the erythrocyte membrane. This channel is activated by oxidative stress,[79] so the generation of a highly reactive singlet oxygen triggers transport through this channel. The channel is permeable to calcium.[80] Hence a coupling of the non-selective cation channel and the Gardos channel could occur as proposed by Kaestner and Bernhardt.[81] The Gardos channel would then realise the potassium efflux, which is seen after PDT of erythrocytes.[76,82] Furthermore, calcium entry into erythrocytes would activate phospolipid scramblase and led to a disruption of the asymmetrical phospholipid distribution.[83,84] This process might be the reason of the observed aggregation of erythrocytes after PDT.[85]
Additionally, the activation of non-selective cation channels leads to sodium influx and hence to swelling of the erythrocytes. This swelling, in connection with the altered membrane stability due to the asymmetrical phospholipid distribution breakdown, might be the cause of haemolysis of the erythrocytes. Haemolysis is well described by a so called ‘colloid-osmotic’ model.[86–88] The phenomenology of the kinetics of this process has been reviewed by Grossweiner.[89] Activation of the non-selective cation channel causes, beside breakdown of phosphatidylserine asymmetry, membrane blebbing.[90,91] These symptoms may be indications of apoptotic death. This is in agreement with a proposed ‘suicidal mechanism’ for red blood cells.[81] The mechanism is summarised in Fig. 5.
Erythrocytes were believed to be a good cell model for screening new potential dyes for PDT.[92] A phototoxicity test based on the use of erythrocytes (Photo-RBC test) was developed by Beiersdorf AG.[93] This test is based on photohaemolysis as well as on haemoglobin oxidation. However, recent studies have shown that photosensitisers with similar behaviour in vivo were behaving totally different in erythrocytes, and that dyes, which are efficient in cell cultures, are ineffective in erythrocytes.[19,73] Although, the universality of the photohaemolysis part of the Photo-RBC test must be questioned, the quality of the test lies in the combination of haemolysis with met-haemoglobin formation,[94,95] which is known to occur during extensive exposure to sunlight as a result of photodynamic reactions[96].
Sterilisation of red blood cell concentratesOne promising application of photosensitisation is sterilisation of red blood cell concentrates.[97] Phthalocyanine ‘Pc 4’, a silicon based phthalocyanine {HOSiPc-OSi(CH3)2(CH2)3N(CH3)2} and its derivatives, is one of the sensitisers most widely used for this application.[97] It has been shown that Pc 4 can sensitise photoinactivation of vesicular stomatitis virus,[98] malaria (plasmodium falciparum)[99] and even multiple forms of the human immunodeficiency virus.[100] It has been claimed that the damage to the red blood cells (haemolysis) is just slightly above that of control cells.[101] The problem of enhanced red blood cell aggregation after PDT can be avoided using a mixture of antioxidants, consisting of vitamin E, mannitol and glutathione.[85] Haemolysis of red blood cells after incubation with Pc 4 and exposure to red light depends on a number of experimental parameters like presence of antioxidants, incubation time and delivery vehicle. For example negatively charged liposomes as a delivery vehicle minimise its binding to erythrocytes while not reducing virus inactivation.[102] After the treatment and three weeks of storage the haemolysis can be as high as 43%, but under other conditions less than 0.5%. This is still at least three times higher than that in control samples.[18,103]
A way to protect erythrocytes from haemolysis during PDT is the use of compounds similar in structure to the photosensitiser but that are not photosensitisers under the treatment conditions. For example, the use of quinacrine to prevent binding of dimethyl-methylene blue to erythrocytes.[104]
Another type of protection is the addition of dipyridamole.[105] It inhibits the ion transport across the band 3 protein.[106] Fortunately, virus inactivation is not affected by dipyridamole.[105] Dipyridamole needs to be removed from the blood samples before they can be used for transfusion, since band 3 protein mediates the oxygen transport across the membrane. It is also questionable whether malaria inactivation can be achieved, if the red cells are to be protected.
PDT may affect lymphocytes in a blood sample. One day after PDT with Pc 4, 32% of all types of lympocytes survived, while four days later this percentage had decreased to 8%.[107] These values are similar to what is found for gamma irradiation (20 Gy) of erythrocyte concentrates.[108] Thus, PDT of blood leads to comparable lymphocyte damage as other potential methods.
The problem of bacterial contamination of erythrocyte concentrates and blood components is rising. Thionine, a demethylated derivative of methylene blue, was tested as a photosensitizer for bacterial decontamination. However, PDT using thionine was strongly inhibited in the presence of plasma. This could be overcome when the photodynamic treatment is followed by a small exposure to UV-B light (1 J cm−2).[109] In addition to the above mentioned dyes, a number of other dyes have been investigated for blood sterilisation: benzoporphyrin derivatives,[23] merocyanine 540,[110] methylene violet,[111] pheophorbide derivatives[112] and dimethyl-methylene blue[105,111].
Little is expected to be achieved with respect to improvement of the treatment of EPP from erythrocyte related research. Since the gene encoding for ferrochelatase has been cloned,[113] gene therapy holds particular promise as a future therapy.[114] In mouse models a long time cure by gene therapy[115,116] as well as stem cell therapy[117] has already been successfully performed.
PDT, through its action on erythrocytes, offers an opportunity to block the microvasculature supplying the tumour, and hence might provide a universal method of tumour destruction. However, to achieve selective control remains a problem and is certainly a target for future research. The generation of fluorescent bilirubin upon UV-A irradiation of erythrocytes has little clinical impact but a significant importance in imaging techniques of erythrocytes[118] since a number of convenient fluorophores (e.g. the calcium ionophores indo-1 and fura-2) require UV-A excitation.[119]
As mentioned in the cell model section, human erythrocytes are convenient models for studies of fundamental principles of PDT. A disadvantage is their lack of cell organelles, especially mitochondria, because the metabolism of the mitochondria is essential for ALA-PDT as well as for induction of apoptosis with other forms of PDT.[120] Fortunately, erythrocytes are available from warm-blooded animals, which contain mitochondria. This is the case for avian erythrocytes,[121] which can be conveniently used for relevant PDT studies. The first investigation on photodynamic effects on cell organelles of chicken erythrocytes has been published,[122] although it did not focus on mitochondria.
Apoptosis seems to be a promising field, also when using erythrocytes, because ‘apoptosis like’ behaviour has been detected in human red blood cells, as described in the section ‘Red blood cells as model cells in experimental PDT research’ (Fig. 5). The involvement of this mechanism in photodynamic damage needs to be further explored.
PDT of erythrocyte concentrates aimed at blood sterilisation is still under investigation and waiting for clinical applications. Concerning viral sterilisation, this method has to compete with agents that target the viral nucleic acids by irreversible modification[123,124] that has been tested in animals[125,126] and is already in clinical trials.[127] Nevertheless, blood decontamination by PDT may have a future for in vivo use. In animal experiments chloroquine resistant malaria strains were found to be more sensitive to Pc 4 than a chloroquine-sensitive strain (preliminary results)[99].