1
Erythrocytes—the ‘house elves’ of photodynamic therapy

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

2
Photodynamic therapy (PDT) and fluorescence diagnosis (FD) are being developed for a number of clinical applications.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

3
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).

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

4
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.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met1

5
Thus, the erythrocytes are partly protected while the endothelial cells suffer photodamage.During photodynamic therapy in vivo erythrocytes are regularly photosensitised.

Type: Method | Advantage: Yes | Novelty: None | ConceptID: Met2

6
This side effect is partly intended but mostly unwanted, and a summary of this topic is given.

Type: Method | Advantage: No | Novelty: None | ConceptID: Met3

7
Furthermore, the effect of UV-A on erythrocytes that is accompanied with the formation of bilirubin is reviewed.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj2

8
Erythrocytes serve as convenient model cells for experimental research.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

9
Such use of erythrocytes to screen new photosensitisers may be of limited value.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp1

10
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.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp2

11
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

12
Photodynamic effects on the erythrocytes themselves should be avoided.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

13
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

Introduction

14
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 formed1,2.1O2 can react with and destroy a number of biomolecules,3,4 including lipids, proteins and DNA.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

15
Photodynamic effects in living cells are probably almost as old as the origin of life.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp3

16
The first scientific description of the effect was published by von Tappeiner and Raab in .19005,6

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

17
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 .19958

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

18
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

19
As first described in 1953, an error in the last step of haem synthesis leads to fluorescent erythrocytes.10,11

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

20
This inherited disease is called erythropoietic protoporphyria (EPP) according to the definition of Magnus et al. in .196112

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

21
However, the first documentation of EPP dates back to .192613,14

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

22
Since photodynamic effects in erythrocytes are characteristic for EPP, a section of this paper is dedicated to this disease.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj3

23
Potential possibilities for the use of the photosensitation in therapy were explored in the 1940s.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

24
This research was revived in the mid-seventies, partly due to the advent of lasers and fibre optic systems.15

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

25
Since that time numerous photosensitisers have been studied and tested for both PDT and fluorescence diagnosis (FD).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

26
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

27
(ii) It is believed that the generation of reactive oxygen species, notably singlet oxygen, is the main molecular mechanism of photodynamic therapy.17

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

28
Therefore, oxygen is needed for efficient PDT.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

29
Red blood cells are of particular interest since they constitute the oxygen transport system of the body.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj4

30
Differences in the spectra of oxygenated and deoxygenated haemoglobin can be used to monitor the oxygenation of the tissue under treatment.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

31
(iii) The blood is used to control the sensitiser distribution.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

32
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

33
Liquid chromatography is a convenient method to determine photosensitiser concentrations in blood.18

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

34
(iv) In photodynamic treatment of systemically applied photosensitisers, photodynamic damage to erythrocytes may occur as a side-effect of PDT.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj5

35
This topic is summarised in the section ‘Photodynamic damage to erythrocytes as a side effect of in vivo photodynamic therapy’.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac15

36
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

37
Upon irradiation with UV-A erythrocytes turn fluorescent.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

38
This is most probably due to the formation of bilirubin.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp4

39
The role of bilirubin in PDT is discussed in a separate paragraph.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj6

40
Red blood cells often serve as model cells for in vitro PDT studies.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

41
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac18

42
This topic is discussed in the section ‘Red blood cells as model cells in experimental PDT research’.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj7

43
In the mid-1980s PDT was introduced in the field of virus destruction.20

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac19

44
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac20

45
The need for bacterial decontamination of blood components is rising.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj8

46
Similar as for EPP it is advantageous to avoid photodynamic effects in erythrocytes.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res9

47
Decontamination of red blood cell concentrates is reviewed in the section ‘Sterilisation of red blood cell concentrates’.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj9

48
Finally ideas for further research on photodynamic effects in red blood cells are given.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj10

Erythropoietic protoporphyria

49
The porphyrias are metabolic diseases caused by disorders in the biosynthesis of haem.24

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac21

50
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac22

51
The haem synthesis pathway is well known26 and is taken advantage of in PDT with 5-aminolaevulinic acid (ALA).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac23

52
ALA is a naturally occurring early metabolite in the pathway.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac24

53
When applied exogenously, ALA induces the formation of protoporphyrin IX (PP IX).27

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac25

54
Impaired activity of the enzyme ferrochelatase, which inserts the iron into PP IX, causes EPP.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac26

55
The ferrochelatase activity of EPP patients is decreased by 70 to 90% compared to that of controls.28–30

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac27

56
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac28

57
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac29

58
Although the intracellular PP IX concentration in erythrocytes decreases with cell age,33 photosensitisation may take place under light exposure.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

59
This effect is illustrated in Fig. 1.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

60
It does not lead to haemolysis of erythrocytes but to a light-triggered displacement of protoporphyrin from erythrocytes to endothelial cells.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

61
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.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

62
When exposed to light, the binding site of PP IX is destroyed.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

63
Since erythrocytes in vivo receive light while circulating in the upper dermal capillaries, they are exposed to pulses rather than to continuous irradiation.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod4

64
Therefore the (haemoglobin-) released PP IX can diffuse through the cell membrane while the erythrocytes move through the ‘dark’ part of the circulation.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod5

65
Thus, they get no membrane damage.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod6

66
PP IX is then bound to plasma proteins, like albumin and haemopexin, and further delivered to endothelial cells.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod7

67
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.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod8

68
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res10

69
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac30

70
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 success41–43.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac31

Photodynamic damage to erythrocytes as a side effect of in vivo photodynamic therapy

71
Damage to erythrocytes was reported after PDT with Photofrin II of cat brain in .198744

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac32

72
The erythrocytes appeared in tightly packed aggregates, which many times filled the luminal space completely.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac33

73
Individual erythrocytes of the aggregates occasionally showed morphological evidence of intravascular haemolysis.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac34

74
In 1990 Orenstein et al45. found that the lumen of the vessels were filled with swollen erythrocytes after PDT of chicken comb.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac35

75
These examples show that erythrocytes may be part of a mechanism inducing necrosis in tumours by blocking the vasculature.46–48

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac36

76
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac37

77
The latter mechanism is similar to the first mentioned one.50

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac38

78
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res11

79
These are derivatives of second generation photosensitisers introduced into or attached to chemical devices.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met4

80
This modification increases the biological specificity to deliver photosensitisers to a defined cell type.

Type: Method | Advantage: Yes | Novelty: None | ConceptID: Met5

81
Therefore, photodamage of the erythrocyte during the transit of the photosensitiser can be avoided or at least significantly reduced.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res12

The UV-A induced formation of bilirubin and its photodegradation

82
A photodynamic effect on erythrocytes occurs after UV-A irradiation in vitro: Erythrocytes, when irradiated with UV-A, turn fluorescent.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

83
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).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

84
Due to the biochemical decomposition of haemoglobin51 and the shape of the spectrum52 it seems obvious that the UV-A induced fluorescence in erythrocytes is caused by bilirubin type photoproducts of haemoglobin.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res13

85
There are two well described photodynamic effects of bilirubin:

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac39

86
(i) The photosensitising effect of bilirubin is described for in vitro systems.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac40

87
It is an efficient photosensitiser in general53,54 and especially in erythrocytes.55

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac41

88
UV-A penetrates the skin down to the dermis.56

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac42

89
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res14

90
However, the clinical reports of erythrocyte damage after UV-A irradiation are missing.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac43

91
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.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp5

92
The situation seems to be different when bilirubin is already present in the blood (see below).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res15

93
(ii) In vivo photodegradation of bilirubin (not exclusively by UV-A, more prominent by blue light) is observed in jaundiced newborns.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac44

94
The phototherapy concept for newborns suffering from hyperbilirubinaemia57 is in accordance with Fig. 2 (parts A and B) showing a fast bilirubin photodegradation or conversion in non-fluorescent isoforms.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res16

95
Although the bilirubin in the newborns originates from erythrocytes,57 the photodynamic effect on erythrocytes is limited.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res17

96
However, it was shown that bilirubin is bound by human erythrocytes.58

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac45

97
There are clinical reports of photodynamic damage on erythrocytes after phototherapy.59,60

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac46

98
Such damage, namely the osmotic fragility, was confirmed in an animal model61 as well as in vitro.62

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac47

99
Bruzell63 recently reviewed the role of bilirubin in jaundice.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac48

Red blood cells as model cells in experimental PDT research

100
Erythrocytes are popular model cells since they are easy to obtain in large numbers with minimal consequences for the donor.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac49

101
They are the most easily available living cells from humans.64

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac50

102
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).

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met6

103
Examples are given in Fig. 3 and 4.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

104
Fig. 3 shows data for several tetraphenylporphyrins, which were studied with respect to their ability to accumulate in biological membranes retaining their photophysical properties.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

105
Fluorescence lifetimes in erythrocyte ghosts were measured and compared with the corresponding lifetimes in organic solvents.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met7

106
Part A shows the structural formula of unsubstituted tetraphenylporphyrin, a totally ‘flat’ molecule.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod9

107
In contrast, diethyltetraphenylporphyrin and tetraethyltetraphenylporphyrin (Fig. 3B) have a pronounced non-planar, three-dimensional structure.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

108
The data are derived from X-ray scattering experiments.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met8

109
However, there were no differences in the magnitude of the fluorescence lifetimes (Fig. 3C) in the solvents compared to the ghosts.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res18

110
The differences in fluorescence lifetimes among different dyes are due to a different bending of the dye molecules.65

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac51

111
The long fluorescence lifetimes of dyes incubated in erythrocyte ghosts show that mainly monomers accumulate in the erythrocyte membranes.66

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac52

112
Another application of erythrocyte ghosts as model membranes is related to the generation of singlet oxygen yield during photodynamic action.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj11

113
The generation of singlet oxygen during PDT is known at least since the 1960s.67,68

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac53

114
Later it was also shown to occur in red blood cells.69

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac54

115
Oelckers et al. were able to detect singlet oxygen luminescence from the inside of a membrane in erythrocyte ghosts.70

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac55

116
This was the first demonstration ever of singlet oxygen luminescence originating from inside a native membrane.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res19

117
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).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

118
Benzoporphyrin derivatives as well as zinc phthalocyanines are effective photosensitizers in cell cultures.71,72

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac56

119
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac57

120
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.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met9

121
However, such measurements are easy to carry out in erythrocyte ghosts.

Type: Method | Advantage: Yes | Novelty: None | ConceptID: Met10

122
Accumulation of the photosensitiser during incubation can be followed in erythrocyte ghosts and cell survival can be monitored by measuring haemolysis (Fig. 4C).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res20

123
However, this practice is controversial.75

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac58

124
Photodynamic damage to erythrocyte membranes starts with potassium release.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod10

125
Haemolysis just indicates disruption of the cell membrane.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod11

126
Ball et al76. found that potassium release and haemoglobin release are just time shifted with respect to each other.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac59

127
This indicates that these processes are causally connected.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac60

128
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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res21

129
Spectroscopic measurements of haemoglobin are much easier and faster to carry out than cell staining and counting.

Type: Method | Advantage: Yes | Novelty: None | ConceptID: Met11

130
This is another advantage of using red blood cells for this type of investigations.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res22

131
The mechanisms behind the above mentioned release of potassium prior to erythrocyte haemolysis are unknown.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res23

132
However, we propose a mechanism related to an operation of a non-selective cation channel78 in the erythrocyte membrane.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod12

133
This channel is activated by oxidative stress,79 so the generation of a highly reactive singlet oxygen triggers transport through this channel.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod13

134
The channel is permeable to calcium.80

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod14

135
Hence a coupling of the non-selective cation channel and the Gardos channel could occur as proposed by Kaestner and Bernhardt.81

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod15

136
The Gardos channel would then realise the potassium efflux, which is seen after PDT of erythrocytes.76,82

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod16

137
Furthermore, calcium entry into erythrocytes would activate phospolipid scramblase and led to a disruption of the asymmetrical phospholipid distribution.83,84

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod17

138
This process might be the reason of the observed aggregation of erythrocytes after PDT.85

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod18

139
Additionally, the activation of non-selective cation channels leads to sodium influx and hence to swelling of the erythrocytes.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod19

140
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.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp6

141
Haemolysis is well described by a so called ‘colloid-osmotic’ model.86–88

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac61

142
The phenomenology of the kinetics of this process has been reviewed by Grossweiner.89

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac62

143
Activation of the non-selective cation channel causes, beside breakdown of phosphatidylserine asymmetry, membrane blebbing.90,91

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod20

144
These symptoms may be indications of apoptotic death.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp7

145
This is in agreement with a proposed ‘suicidal mechanism’ for red blood cells.81

Type: Result | Advantage: None | Novelty: None | ConceptID: Res23

146
The mechanism is summarised in Fig. 5.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod21

147
Erythrocytes were believed to be a good cell model for screening new potential dyes for PDT.92

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac63

148
A phototoxicity test based on the use of erythrocytes (Photo-RBC test) was developed by Beiersdorf AG.93

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac64

149
This test is based on photohaemolysis as well as on haemoglobin oxidation.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met12

150
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac65

151
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 reactions96.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac66

Sterilisation of red blood cell concentrates

152
One promising application of photosensitisation is sterilisation of red blood cell concentrates.97

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot3

153
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac67

154
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac68

155
It has been claimed that the damage to the red blood cells (haemolysis) is just slightly above that of control cells.101

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac69

156
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac70

157
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.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac71

158
For example negatively charged liposomes as a delivery vehicle minimise its binding to erythrocytes while not reducing virus inactivation.102

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac72

159
After the treatment and three weeks of storage the haemolysis can be as high as 43%, but under other conditions less than 0.5%.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac73

160
This is still at least three times higher than that in control samples.18,103

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac74

161
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.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met13

162
For example, the use of quinacrine to prevent binding of dimethyl-methylene blue to erythrocytes.104

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac75

163
Another type of protection is the addition of dipyridamole.105

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met14

164
It inhibits the ion transport across the band 3 protein.106

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod22

165
Fortunately, virus inactivation is not affected by dipyridamole.105

Type: Method | Advantage: Yes | Novelty: Old | ConceptID: Met14

166
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.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met15

167
It is also questionable whether malaria inactivation can be achieved, if the red cells are to be protected.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp8

168
PDT may affect lymphocytes in a blood sample.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp9

169
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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac76

170
These values are similar to what is found for gamma irradiation (20 Gy) of erythrocyte concentrates.108

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac77

171
Thus, PDT of blood leads to comparable lymphocyte damage as other potential methods.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res24

172
The problem of bacterial contamination of erythrocyte concentrates and blood components is rising.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot4

173
Thionine, a demethylated derivative of methylene blue, was tested as a photosensitizer for bacterial decontamination.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac78

174
However, PDT using thionine was strongly inhibited in the presence of plasma.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac79

175
This could be overcome when the photodynamic treatment is followed by a small exposure to UV-B light (1 J cm−2).109

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac80

176
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 derivatives112 and dimethyl-methylene blue105,111.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac81

Future aspects

177
Little is expected to be achieved with respect to improvement of the treatment of EPP from erythrocyte related research.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

178
Since the gene encoding for ferrochelatase has been cloned,113 gene therapy holds particular promise as a future therapy.114

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac82

179
In mouse models a long time cure by gene therapy115,116 as well as stem cell therapy117 has already been successfully performed.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac83

180
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.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

181
However, to achieve selective control remains a problem and is certainly a target for future research.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot5

182
The generation of fluorescent bilirubin upon UV-A irradiation of erythrocytes has little clinical impact but a significant importance in imaging techniques of erythrocytes118 since a number of convenient fluorophores (e.g. the calcium ionophores indo-1 and fura-2) require UV-A excitation.119

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac84

183
As mentioned in the cell model section, human erythrocytes are convenient models for studies of fundamental principles of PDT.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

184
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

Type: Method | Advantage: No | Novelty: None | ConceptID: Met16

185
Fortunately, erythrocytes are available from warm-blooded animals, which contain mitochondria.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac85

186
This is the case for avian erythrocytes,121 which can be conveniently used for relevant PDT studies.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac86

187
The first investigation on photodynamic effects on cell organelles of chicken erythrocytes has been published,122 although it did not focus on mitochondria.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac87

188
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).

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

189
The involvement of this mechanism in photodynamic damage needs to be further explored.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot6

190
PDT of erythrocyte concentrates aimed at blood sterilisation is still under investigation and waiting for clinical applications.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac88

191
Concerning viral sterilisation, this method has to compete with agents that target the viral nucleic acids by irreversible modification123,124 that has been tested in animals125,126 and is already in clinical trials.127

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac89

192
Nevertheless, blood decontamination by PDT may have a future for in vivo use.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

193
In animal experiments chloroquine resistant malaria strains were found to be more sensitive to Pc 4 than a chloroquine-sensitive strain (preliminary results)99.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac90