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ChapterElectron Transfer-Supported
Photodynamic Therapy
Kazutaka Hirakawa

Abstract
Photodynamic therapy (PDT) is a less-invasive treatment of cancer and precancerous lesions. Porphyrin derivatives have been used and studied as the photosensitizers for PDT. In general, the biomacromolecules oxidation by singlet oxygen, which is
produced through energy transfer from the photoexcited photosensitizers to oxygen
molecules, is an important mechanism of PDT. However, the traditional PDT effectmay be restricted, because tumors are in a hypoxic condition and in certain cases,
PDT enhances hypoxia via vascular damage. To solve this problem, the electron transfer-mediated oxidation of biomolecules has been proposed as the PDT mechanism.
Specifically, porphyrin phosphorus(V) complexes demonstrate relatively strong photooxidative activity in protein damage through electron transfer. Furthermore, otherphotosensitizers, e.g., cationic free-base porphyrins, can oxidize biomolecules throughelectron transfer. The electron transfer-supported PDT may play the important roles
in hypoxia cancer therapy. Furthermore, the electron transfer-supported mechanism
may contribute to antimicrobial PDT. In this chapter, recent topics about the biomolecules photooxidation by electron transfer-supported mechanism are reviewed.
Keywords: Photoinduced electron transfer, porphyrin phosphorus(V) complex,
protein oxidation, cationic porphyrin, phenothiazine dyes

1. Introduction
Photodynamic therapy (PDT) is a less-invasive treatment of cancer and othernonmalignant conditions [1–3]. This treatment is a medicinal application of photochemistry. Antimicrobial treatment, called as antimicrobial photodynamic therapy(aPDT) or photodynamic antimicrobial chemotherapy (PACT), is also important
application [4–7]. In the case of cancer treatment, less-toxic PDT reagents, photosensitizers, cause oxidative damage to biomolecules, including protein, nucleic
acids, and/or other compounds, under visible-light irradiation. This photosensitizedreaction results in necrosis or apoptosis of cancer cells [1–3]. As the PDT photosensitizers, porphyrins have been extensively studied and used [8–11]. For example, porfimer sodium [12, 13] and talaporfin sodium [13], an oligomer and a monomer of afree-base anionic porphyrin, respectively, are well-known photosensitizers in clinicaluse. In general, the porphyrin photosensitizer (e.g., almost 60 mg/body for talaporfin
sodium) is given for the target tissue, followed by irradiation of the visible light (e.g.,
664 nm, 150 mW cm−2, and 10 J cm−2). To reduce the risk of adverse side effects,
the development of efficient photosensitizers that work with harmless weak lightis important. Furthermore, consideration of PDT mechanism is also important todevelop effective photosensitizer. Most of porphyrins have relatively large quantum1

Photodynamic Therapy - From Basic Science to Clinical Researchyield (ΦΔ) for singlet oxygen (1O2), a reactive oxygen species (ROS), generation [14].
O2 can be easily generated by relatively small energy photon of long wavelength
visible light and/or near infrared radiation (wavelength ≳ 770 nm) through energy
transfer from photoexcited photosensitizer to oxygen molecule [15–17]. Radiation in
the long wavelength region called “optical window”, 600 ~ 1300 nm, can penetratehuman tissue deeply [18]. Therefore, 1O2 is the important reactive species of porphyrin-based PDT. However, the phototoxic effect of 1O2 on PDT is restricted because of
the hypoxic condition of tumors [19–22]. Furthermore, in certain cases, PDT itself
enhances hypoxia [23] via vascular damage [24]. This “hypoxia problem” of tumor isvery important to improve the PDT effect.
Oxidation is defined as the oxygenation, hydrogen extraction, and electron
extraction. Electron extraction from biomolecules to photoexcited photosensitizer
is also the mechanism of oxidative biomolecule damage. This electron transferoxidation may be an important mechanism to resolve the “hypoxia problem” and todevelop the effective PDT photosensitizers. Phosphorus(V) porphyrins [25, 26] andcationic free-base porphyrins [27] have relatively strong oxidative activity throughelectron transfer [28]. Furthermore, electron transfer process can be control bysurroundings condition, for example pH of medium [29, 30].
In this chapter, recent studies about the electron transfer-supported photosensitizer for PDT are reviewed. The examples of activity control of photosensitizer forthe cancer-selective PDT are also introduced. In the last section, the role of electrontransfer mechanism in aPDT is discussed.
1

2. Electron transfer oxidation as a mechanism of photosensitizedbiomolecule damage
In general, photosensitized biomolecule damage can be explained by oxygenindependent mechanism (Type I mechanism) and oxygen-mediated mechanism(Type II mechanism) (Figure 1) [31–33]. Because the electron transfer-mediatedbiomolecule oxidation does not absolutely require oxygen, this mechanism iscategorized as Type I mechanism. On the other hand, biomolecule oxidation
through 1O2 generation is defined as Type II mechanism (Type II, major). Another
ROS-mediated process, superoxide (O2•−)-mediated biomolecule oxidation is also
categorized as the Type II mechanism (Type II, minor). Although O2•− is produced
through electron transfer from photoexcited photosensitizer, it’s not categorized as
the Type I mechanism. The initial process of electron transfer-mediated biomolecule oxidation is an electron extraction from the targeting biomolecule, such asprotein, to the photoexcited photosensitizer.
2.1 Driving force dependence of electron transfer
The driving force of electron transfer, Gibbs energy (ΔG), is determined by
the excitation energy of photosensitizer (photon energy) and the redox potentialof photosensitizer and targeting biomolecule. The electron transfer is a relaxation
process of photoexcited photosensitizer. Fast electron transfer is advantageous foran efficient electron transfer. Due to the Marcus theory [34, 35], the rate constant ofelectron transfer (kET) is expressed using ΔG as follows:2- ( DG + l )4p 3,
VDA 2 exp
kET = 24 l KBTh l K BT*

2

(1)

Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

Figure 1.
Relaxation process of photoexcited state of photosensitizer and the typical photosensitized biomoleculedamaging mechanisms.

where h is Plank constant, λ is the reorganization energy, KB is the Boltzmann
constant, and VDA is the effective electronic Hamiltonian matrix element. The λ canbe calculated from the following equation:l=

1 2 öæ 1 1 ö
e2 æ 1++ ÷ ç - ÷,ç
4pe 0 è 2rD 2rA d ø è n2 e ø(2)where e is the elementary charge, ε0 is the vacuum permeability(8.854 × 10−12 F m−1), rD and rA are the radius of the electron donor and that ofacceptor, respectively, d is the distance between electron donor and acceptor, n is
the refractive index, and ε is the static dielectric constant of surrounding material.
Since the VDA is determined by the overlap between wavefunctions of electron
donor and acceptor, the electron transfer rate strongly depends on the d, anddecreased exponentially with an increase in d. Therefore, association between
photosensitizer and targeting biomolecule is very important. The ΔG, driving forceof electron transfer, is expressed as follows:
DG = e ( Ered – Eox ) – E0–0 ,(3)

where Ered is the redox potential of a one-electron reduction of photosensitizer,
Eox is the redox potential of a one-electron oxidation of targeting biomolecule, andE0–0 is the 0–0 energy (singlet excited (S1) energy) of photosensitizer. The Eq. (1)indicates that kET becomes maximum at ΔG = λ. However, in general, large -ΔG is3

Photodynamic Therapy - From Basic Science to Clinical Researchadvantageous for fast electron transfer. Therefore, small (small absolute value) Ered
and/or large (large absolute value) Eox is appropriate for effective electron transfer.
To evaluate the electron transfer in the triplet excited (T1) state, the “E0–0” term in
Eq. (3) is replaced with the T1 state energy. Because T1 state energy is smaller thanE0–0, in general, electron transfer oxidation by T1 state photosensitizer becomesdifficult.
2.2 Excitation energy and electron transfer
Excitation energy (photon energy) strongly affects the electron transfer rate andefficiency as the Eq. (3). Indeed, an ultraviolet photosensitizer can oxidize DNA,
which is relatively resistant to the electron extraction, through photoinduced electron transfer [32, 33]. However, ultraviolet radiation is harmful for human tissue.
Furthermore, long wavelength visible light or near infrared radiation can penetratehuman tissue deeply as mentioned above as the optical window [18]. Therefore,
visible light (or near infrared) photosensitizer, such as porphyrins and phthalocyanines, are important for PDT. To realize the electron transfer photosensitizer, which
can be excited by long wavelength light, the design and synthesis of photosensitizer
molecules with small Ered value are required. However, a molecule with small Ered
has tend to decay through reduction by surrounding molecules, and small Ered is not
appropriate for stability of molecule.
2.3 Kinetics of electron transfer
In general, electron transfer can be demonstrated by a transient absorption spectrum measurement [36, 37] and a time-resolved electron paramagnetic resonancemeasurement [38, 39]. The kET values can be determined by the analysis of transientabsorption spectra. Fluorescence lifetime measurement is also an important method[40]. Although fluorescence lifetime is affected by various factors other than electron transfer, it is sensitive and convenient method. If other factors can be excluded,
this method is advantageous for the kinetic evaluation of electron transfer. The kET
value can be obtained using fluorescence lifetime by the following equation:

kET =

1

tf

-

1

t f0

,

(4)

where τf is the observed fluorescence lifetime of photosensitizer with electron
donor (targeting biomolecule) and τf0 is that without electron donor. In general,kET becomes larger than 108 ~ 109 s−1 in the case of electron transfer in the S1 state,because lifetime of most of porphyrin S1 state is order of several nanosecond. In thecase of T1 state, the lifetime is order of microsecond and the rate constant becomes
relatively small. As mentioned above, the T1 state is not appropriate for electrontransfer oxidation from the thermodynamic point of view.

3. Phosphorus(V) porphyrin photosensitizer
Porphyrin derivatives have been used as clinical photosensitizer for PDT [8–11].
Porfimer sodium [12, 13] and Talaporfin sodium [13] are famous examples of clinically used photosensitizers. The PDT mechanism of these porphyrins is 1O2 generation. The photochemical property of porphyrin can be changed by the replacementof the central atom and substitution. It has been reported that phosphorus(V)4Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

porphyrin can oxidize biomolecules, such as nucleobase [41], protein [42–48], andother biomolecules [49, 50] through electron transfer.
3.1 General property of phosphorus(V) porphyrin
General procedure of synthesis method of phosphorus(V) porphyrin is a
reflux of free base porphyrin with phosphoryl chloride in dry pyridine [51]. The
photochemical property of phosphorus(V) porphyrin can be improved by thesubstitution of the meso- or β-positions and the axial ligand (Figure 2) [42–53]. An
example of phosphorus(V) porphyrin, diethoxyP(V)tetrakis(4-methoxyphenyl)
porphyrin chloride, is shown in Figure 3. The calculation with density functional theory (DFT) at ωB97X-D/6-31G* level shows the distorted structure ofphosphorus(V) porphyrin. Their distorted structures have been reported fromthe results of X-ray crystal analysis [54]. Phosphorus(V) porphyrins introduced inthis chapter are listed in Table 1. Because phosphorus(V) porphyrin is a cationic

Figure 2.
Structures of phosphorus(V) porphyrins.

Figure 3.
Optimized structure of Por10 by the DFT calculation at ωB97X-D/6-31G* level.
5

Photodynamic Therapy - From Basic Science to Clinical Researchporphyrin, its water solubility is relatively large. Furthermore, hydrophilic substitution markedly increases the water solubility [55]. One of the most importantcharacteristics of phosphorus(V) porphyrin is relatively small Ered value dueto the positive charge of the central phosphorus atom, resulting in the strongoxidative activity in the photoexcited state. This character is very important aselectron transfer-supported photosensitizer for PDT. Furthermore, in general,phosphorus(V) porphyrin has relatively large quantum yield of photosensitized
1
O2 generation in an aqueous solution (ΦΔ is more than 0.5, Table 1) due to theeffective intersystem crossing [42–47]. In the presence of enough oxygen molecules, phosphorus(V) porphyrin can oxidize biomolecule through 1O2 generation,a traditional PDT mechanism.
3.2 Photosensitized protein damage by phosphorus(V) porphyrin throughelectron transfer
Isolated amino acids, a water-soluble protein, and enzymes have been usedas the targeting biomacromolecules to examine photosensitizer activity ofphosphorus(V) porphyrins [42]. For example, human serum albumin (HSA),a water-soluble protein, is a convenient target. The crystal structure and aminoacid sequence of HSA have been clarified [56]. In addition, HSA has major drug
specific binding sites identified as Sudlow’s site I and site II [57]. The mono-cationicphosphorus(V) porphyrins listed in Table 1 are well-soluble in organic solvents(e.g., alcohol) rather than water, indicating the hydrophobic character beside thehydrophilicity. Therefore, binding interaction between HSA and phosphorus(V)Compounds

Ered / V

E0–0 / eV

Por1

−0.30

2.04, PBS1.25EtOH

Por2

a

2.03, PBS1.25EtOHa
b

−0.50

2.03, PBS

Φf

0.017, PBS2.5EtOHc
c0.023, EtOH

Ref.

ΦΔ
0.96, EtOH

[42]

b

0.64, PBS
0.93, PBS2.5EtOHc[42]a,[43]b,[52]c

Por3−0.54e2.04, PBSe

0.59, PBSd[44]d,[50]e

Por4−0.40e2.03, PBSe

0.68, PBSd[44]d,
[50]e

Por5

−0.51

2.03, PBS

0.048, PBS

0.88, PBS

[45]

Por6

−0.51

2.03, PBS

0.043, PBS

0.80, PBS

[45]

Por7

−0.54

2.02, PBS1.25EtOH

0.94, EtOH

[42]

Por8

−0.33

0.029, PBS

0.97, PBS

[46]

Por9

−0.58

0.024, PBS

0.86, PBS

[46]

Por10

−0.58

1.96, PBS1.0EtOH

0.067, EtOH

0.84, EtOH

[47]

Por11

−0.43

1.98, PBS1.0EtOH

0.086, EtOH

0.82, EtOH

[47]

Por12

−0.57

0.029, PBS

0.83, PBS

[46]

Por13

−0.55

2.01, PBS1.0EtOH

pH-dependent

pH-dependent[48]

2.00, PBS2.5EtOH

0.034, EtOH

ND, PBS2.5EtOH

[52]

Por14

Ered: measured in acetonitrile (vs. saturated calomel electrode; SCE), PBS: 10 mM sodium phosphate buffer(pH 7.6) solution, EtOH: ethanol, PBSETOH2.5: PBS containing 2.5% ethanol, PBSETOH1.25: PBS containing 1.25%
ethanol, PBSETOH1.0: PBS containing 1.0% ethanol, Φf: Fluorescence quantum yield. ND: not detected.

Table 1.
Examples of phosphorus(V) porphyrin photosensitizers and their photochemical properties.
6Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

porphyrins is expected and their binding site can be speculated. Because theelectron transfer-mediated oxidation strongly depends on the distance between
photosensitizer and the target molecule, a binding interaction is very important.
HSA has one tryptophan, which is easily oxidized by oxidative stress, including 1O2
and electron transfer reaction [42–47, 58]. Tryptophan can emit relatively strong
fluorescence and its damage can be detected by fluorescence measurement [45, 58].
Using these characteristics of HSA, the oxidative damage of tryptophan residue by
photosensitized reaction can be easily examined by a fluorometry [45–47, 58].
Qualitative study of HSA photodamage by phosphorus(V) porphyrins wasreported using Por2 [43]. Por2 oxidized the tryptophan of HSA through 1O2
generation and electron transfer. It has been considered that damaged tryptophan
is changed to N-formylkynurenine and other decomposed products [59, 60]. 1O2
can oxidize the tryptophan residue of HSA [61]. Using isolated amino acids, it has
been demonstrated that tyrosine and tryptophan can be oxidized by photoexcited
Por2 [42].
Photosensitized HSA damage by Por5 and Por6 was quantitatively clarified [45].
Por5 and Por6 bound to HSA and damaged its tryptophan residue during photoirradiation. Por5 and Por6 photosensitized 1O2 generation, and the contribution of
1O2 was confirmed by the inhibitory effect of a 1O2 quencher, sodium azide (NaN3,[62]). From the kinetic analysis, the contribution of electron transfer mechanism to
HSA damage was demonstrated [45]. Fluorescence lifetime measurement and thecalculation of ΔG supported the electron transfer mechanism.
To realize the effective PDT photosensitizer, response of photosensitizers tolong wavelength visible light or near infrared region is important. To improve theabovementioned phosphorus(V) porphyrins, Por5 and Por6, meso-phenyl substituted derivatives were designed and synthesized [46]. Por8, Por9, and Por12
can be excited under the irradiation of long-wavelength visible light (> 630 nm).
These phosphorus(V) porphyrins induced tryptophan oxidation in HSA underillumination with light-emitting diode (central wavelength: 659 nm), and thisprotein photodamage was barely inhibited by NaN3 [46]. Fluorescence lifetimes ofphosphorus(V) porphyrins was decreased by HSA, suggesting the electron transfer
quenching. The ΔG value of electron transfer from tryptophan to the S1 state ofthese porphyrins calculated from their redox potentials also supported the electrontransfer-mediated oxidation.
3.3 Cancer selective photodynamic action of phosphorus(V) porphyrin
photosensitizers
Above mentioned phosphorus(V) porphyrins, Por8, Por9, and Por12,
exhibited the cancer cell selective toxicity under visible light irradiation [46].

Figure 4.
Structures of Por8, Por9, and Por12, and their IC50 values for HeLa cells under photoirradiation [46].
7

Photodynamic Therapy - From Basic Science to Clinical Research

Photocytotoxicity to HeLa cells by these porphyrins are the following order:
Por9 > Por12 > Por8 in the condition of previous report (Figure 4) [46].
Although the half maximal inhibitory concentration (IC50) value for Por8 islargest (least phototoxicity) in the three phosphorus(V) porphyrins, its photocytotoxicity to cancer cells is sufficiently high. Furthermore, Por8 did notexhibit photocytotoxicity to HaCaT cells, cultured human skin cells (normal
cell model). Por9 and Por12 exhibited phototoxicity to HaCaT cells, however,these IC50 value were significantly larger than those for HeLa cells and cellular
DNA damage in HaCat cells were not observed. These three phosphorus(V)porphyrins demonstrated significant PDT effects on mice tumor models [46].
The observed PDT effects by these porphyrins are almost the same, and arecomparable with that of talaporfin sodium. These results suggest the cancer
selectivity of Por8, Por9, and Por12, and lower carcinogenic risk to normal
cells. Specifically, Por8, of which the redox potential is most advantageous forthe electron transfer-mediated biomolecule oxidation, demonstrated the highest
cancer-selectivity and significant PDT effect under irradiation with longwavelength visible light.
3.4 Photoinduced electron transfer by phosphorus(V) porphyrin triggers thechain reaction for NADH decomposition
The electron transfer mechanism can contribute to oxidation other various biomolecules. For example, nicotinamide adenine dinucleotide (NADH), an important
endogenous reductant, becomes an important targeting molecule [50]. The S1 statesof Por3 and Por4 easily extract electron from NADH, resulting in the formation of

Figure 5.
Structures of NADH and its oxidized form, and the electron transfer-triggered chain reaction of NADH
decomposition.
8Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

NAD•, a radical. Further oxidation leads to the irreversible decomposition of NADH
to NAD+ (Figure 5). The total quantum yield of NADH decomposition (ΦD) isexpressed as follows:
F D = F ET ´ F FR ,(5)

where ΦET is the quantum yield of the initial process (electron transfer) and ΦFR is
that of the further reaction to form NAD+. Analysis of the quantum yields, obtained
values of ΦFR became much larger than unity. These findings suggest that the electron
accepting by the photoexcited Por3 and Por4 triggers a chain reaction of NADH oxidation (Figure 5). The initial electron transfer to photoexcited Por3 or Por4 producesNAD•. The NAD• immediately reacts with molecular oxygen to produce O2•−:NAD• + O2 ® NAD+ + O2• - .

(6)

In the following process, O2•− oxidizes NADH and hydrogen peroxide (H2O2) is
produced [63]:NADH + O2• - + H + ® NAD• + H2O2 .

(7)

The electron transfer-mediated reaction induces the chain reaction, resulting
in the acceleration of NADH decomposition and secondary generation of reactive
oxygen species. In the case of direct photosensitized reaction, ultraviolet photon isrequired to produce H2O2 [28]. The secondary formed H2O2 may produce hydroxyl
radicals (•OH), very strong ROS. These results suggest that electron transfer
reaction with visible light irradiation induces a severe toxic effect through a chain
reaction and the formation of H2O2, similarly to the ultraviolet radiation.
3.5 Photosensitized oxidation of folic acid by phosphorus(V) porphyrinthrough electron transfer
Folic acid, a vitamin, is also oxidized through photoinduced electron transfer[64]. Because the fluorescence intensity of folic acid is significantly increasedby the decomposition, a fluorometry of folic acid can be used as a convenient
indicator to evaluate the photosensitizer activities [65, 66]. For example, photosensitized decomposition of folic acid by Por2 through electron transfer was
reported [49]. Photoexcited porphyrin can produce 1O2, and folic acid is alsooxidized by 1O2. The contribution of 1O2-mediated decomposition can be excludedby the effect of 1O2 quencher and the effect of electron transfer reaction can beevaluated.

4. Contribution of the electron transfer mechanism in photosensitizedreaction by cationic porphyrins
Photooxidation activity through electron transfer depends on the redox potential. It has been demonstrated that photoexcited hematoporphyrin, a free baseporphyrin, induces the oxidative electron transfer from the tryptophan residueof bovine serum albumin [67, 68]. Cationic porphyrins show relatively small Eredvalues due to their positive charge. In this section, several examples of electrontransfer-mediated oxidation of biomolecules by cationic porphyrins.
9

Photodynamic Therapy - From Basic Science to Clinical Research

Figure 6.
Structures of H2TMPyP and ZnTMPyP (A), their binding interaction with DNA (B), and the electron transfer
reactions (C). ABG: Amino benzoyl-L-glutamic acid.

4.1 Protein photooxidation through electron transfer by cationic porphyrins
The photosensitized protein damage by tetrakis(N-methyl-p-pyridinio)
porphyrin (H2TMPyP, Figure 6) and its zinc complex (ZnTMPyP, Figure 6)
was reported [69]. Photosensitized reaction of H2TMPyP has been extensively
studied [14, 70]. Water-solubility of H2TMPyP and its analogues is appropriate
for biological study. Furthermore, electrostatic interaction between these cationicporphyrins and biomacromolecules is considered to enhance the electron transfer
reaction with targeting biomolecules. The ΦΔ value of H2TMPyP is relatively
large [14, 69, 71], and photosensitized biomolecule damage caused by H2TMPyP
through 1O2 generation is generally accepted [70, 72]. However, Ered of H2TMPyP
is relatively small [27], and negative ΔG values for photosensitized oxidation ofseveral amino acids through electron transfer are estimated. Therefore, electrontransfer-mediated photooxidation of biomolecules is expected.
H2TMPyP and ZnTMPyP bound to HSA and caused photosensitized oxidationof the tryptophan residue [69]. Three amino acids–tryptophan, phenylalanine, andtyrosine–were also used as target biomolecules, and tryptophan and tyrosine were
photodamaged by these cationic porphyrins. However, H2TMPyP and ZnTMPyP
could not photosensitize the damage of phenylalanine. The protein damage (oxidation of the tryptophan residue) was enhanced in deuterium oxide and inhibitedby NaN3. Analysis of the scavenger effect showed that the absolute quantum yields
of electron transfer-mediated oxidation are 5.3 × 10−3 and 4.0 × 10−3 for H2TMPyP
10

Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

and ZnTMPyP, respectively. The Ered of H2TMPyP (−0.23 V vs. SCE) [27] is lower
than that of ZnTMPyP (−0.85 V) [73]. The values of -ΔG for electron transfer fromtryptophan to their S1 states suggest that H2TMPyP (−1.03 eV) is more oxidative
than ZnTMPyP (−0.53 eV). The estimated value of kET estimated from the fluorescence lifetime for H2TMPyP was 1.0 × 108 s−1. On the other hand, the fluorescence
lifetime of ZnTMPyP was not affected by the interaction with HSA in the presented
experimental condition. Because of the relatively shorter fluorescence lifetimeof ZnTMPyP (1.3 ns), the estimation of kET may be difficult by the fluorescence
lifetime measurement. Furthermore, protein photodamage by the T1 states of
H2TMPyP and ZnTMPyP were also discussed [69]. The lifetimes of their T1 states
are relatively long: H2TMPyP (2.1 μs) and ZnTMPyP (2.7 μs), suggesting that theelectron transfer in the T1 state is kinetically advantageous. The estimated -ΔG ofthe electron transfer from tryptophan to their T1 states (−0.65 eV for H2TMPyP
and − 0.15 eV for ZnTMPyP) suggests that this electron transfer is also possible interms of energy.
4.2 Electron transfer from DNA to photoexcited cationic porphyrins andmicroenvironmental effect of DNA on photoinduced electron transfer
Photoinduced electron transfer between DNA and the cationic porphyrins,
H2TMPyP and ZnTMPyP, was analyzed by the fluorescence measurements(Figure 6) [74]. Absorption spectrum and circular dichroism measurements
showed that H2TMPyP mainly intercalates to calf thymus DNA, whereas
ZnTMPyP binds into a DNA groove. An electrostatic interaction with DNA raises
their redox potentials of the binding cationic porphyrins. In the presence of DNA,
the fluorescence intensity of these porphyrins was almost the same as that without
DNA. The Eox of H2TMPyP (>1.30 V vs. SCE in water) [27], ZnTMPyP (1.18 V vs.
SCE in water) [73], and guanine (1.24 V vs. SCE in acetonitrile) [75, 76] suggested that electron transfer by the S1 state of H2TMPyP is possible in terms ofenergy. Furthermore, the electron donating character of guanines increased in
the double-stranded structure [77–79]. However, the fluorescence measurements
indicated that the S1 states of these porphyrins are barely quenched by DNA.
These results could be explained by that an electrostatic interaction between
cationic porphyrins and an anionic DNA strand should increase the redox potential of porphyrins, leading to the inhibition of the electron transfer. In the casesof their higher excited states, secondary excited singlet (S2) states, the electrontransfer from DNA was observed. The lifetime of S2 state is significantly short (afew picoseconds). However, the Ered value of their S2 states are large (larger Eredvalue of the excited state indicates stronger oxidative activity); >2.14 V vs. SCE for
H2TMPyP and 1.94 V vs. SCE for ZnTMPyP. Therefore, the S2 states of porphyrins
are thermodynamically strong oxidants through electron transfer mechanism.
Photoinduced electron transfer from these porphyrins to benzoquinones,
electron acceptors, and that from N-(4-aminobenzoyl)-L-glutamic acid (ABG), an
electron donor, to these porphyrins were also studied [74]. As mentioned above, theelectrostatic interaction with DNA raises the redox potential of cationic porphyrins(i.e. decreases the oxidative property of cationic porphyrins). Therefore, the DNA
microenvironment inhibited the electron transfer from ABG, an electron-donating
quencher, to the binding porphyrins. On the other hand, the electron transfer fromthe binding porphyrins to benzoquinones, an electron-accepting quencher, wasenhanced. A steric effect by the DNA strand was also important. A hydrophobic
bulky electron acceptors forms stacking complex with porphyrins, resulting in thestrong fluorescence quenching. The interaction with DNA strand cleaves this stacking interaction and inhibit the electron transfer to the benzoquinone. In summary,
11

Photodynamic Therapy - From Basic Science to Clinical Research

the DNA microenvironment significantly affects the electron transfer property of thebinding cationic porphyrins through an electrostatic interaction and the steric effect.

5. Activity control based on the electron transfer
Electron transfer can be controlled by the surrounding environment. For example, pH is an important factor to control the photoinduced electron transfer [29, 30,
48, 80, 81]. Since it has been reported that cancer cells are slightly acidic (pH 6 ~ 7)
against normal tissues (pH 7 ~ 7.4) [82–85], control of the electron transfer of thephotosensitizer by pH can be applied for the development of cancer-selective PDT.
In the cases of pH-dependent 1O2 photosensitizers, the redox control [30, 86–88],
the structure change [89], and the control of intersystem crossing [90] by pH havebeen reported as the important concepts. Several types of pH-activatable-porphyrin
photosensitizers [30, 88], including a phosphorus(V) porphyrin [48, 81], have beenreported. In addition, a self-quenching of the photoexcited molecules can be also
used to control the activity [47]. In this section, several examples about the activity
control of electron transfer-photosensitizers are introduced.
5.1 Electron transfer control by pH
The biomolecule oxidation activity of photosensitizer through electron transfer
can be controlled by using changeable electron donor. Por13 was designed andsynthesized to control the photodynamic activity of phosphorus(V) porphyrin
photosensitizer (Figure 7) [48]. As an electron-donor, 6-methylpyridine was used.
The photoexcited Por13 is quenched through intramolecular electron transfer
and this quenching is suppressed by protonation of the methylpyridine moiety,an electron donor. The pKa of protonated methylpyridine moiety was about 7,
and fluorescence lifetime of Por13 was lengthened under an acidic condition by

Figure 7.
Scheme of the activity control of photosensitizer, Por13, by pH and the relaxation processes of photoexcited state.
12Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

suppression of the quenching through intramolecular electron transfer by methylpyridine. The quantum yields of photosensitized 1O2 generation and biomoleculeoxidation through electron transfer mechanism were also increased under acidic
condition. NADH oxidation by Por13 through photoinduced electron transfer was
successfully enhanced under acidic conditions. However, photosensitized protein
damage (oxidative damage of HSA) through electron transfer was decreased underan acidic condition, and relatively strong protein damage was observed undera neutral condition. It is explained by the fact that a relatively weak association
between protein and Por13 under an acidic condition due to electrostatic repulsion.
Protonated protein under acidic condition decreases the association with cationicporphyrin, resulting in the suppression of the electron transfer from the aminoacids. Furthermore, the hydrophobic environment of protein inhibits the electrontransfer-quenching of Por13. This study shows the difficulty of activity control ofphotosensitizers by pH, because other factors significantly affect the photoinduced
electron transfer.
5.2 Activity control through the self-quenching of photosensitizers
DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrins, Por10 and Por11, analogues of above mentioned Por9, were synthesized [47]. Their water-solubilities
were smaller than that of Por9, and these porphyrins form self-aggregation
complexes (Figure 8). Photoexcited states of Por10 and Por11 were effectively quenched through this aggregation (concentration quenching). These
phosphorus(V) porphyrins can bind to the hydrophobic pocket of HSA, resulting
in dissociation of their self-aggregation states (Figure 8). Calculating simulation
showed the distance between the tryptophan residue and the porphyrin molecules
as follows: 24.4 Å (Por10) and 23.5 Å (Por11). Fluorescence lifetime of theseporphyrins were recovered by the dissociation of self-aggregation. Photoirradiation
to these porphyrins binding to HSA induced the oxidation of tryptophan through1
O2 generation and electron transfer. The axial fluorination of ethoxy chain ofcentral phosphorus atom reduced the Ered of porphyrin ring. The electron transfer

Figure 8.
Scheme of the activity control of photosensitizers, Por10 and Por11, through the self-aggregation andinteraction with HSA.
13

Photodynamic Therapy - From Basic Science to Clinical Researchrate constant from the tryptophan residue of HSA to Por11 is larger than that of
Por10, due to the effect of axial fluorination. The substitution by fluorine, thehighest electronegative element, showed the improving effect on photooxidation ofprotein through electron transfer. However, the fluorination decreased the binding interaction with HSA. In the presence of same concentration of porphyrins,
Por10 exhibits higher damaging activity to HSA under photoirradiation. These
results suggest that selective interaction is important for electron transfer-mediatedphotodamage of biomolecules. These porphyrins demonstrated the photocytotoxicity to HaCaT cells. The IC50 value of Por11 was lower (stronger cytotoxicity) that
Por10. Photooxidative activity of Por11 through electron transfer and enhanced
cellular uptake by the fluorination may play the important role in this photocytotoxic effect. Furthermore, Por10 and Por11 barely induce cellular DNA damage
to HaCaT cells, similarly to Por8, Por9, and Por12. Therefore, their carcinogenic
risks are also small. The self-aggregation of photosensitizers can be used to suppress their photosensitizing activity. These results suggest that the PDT activity ofself-aggregation photosensitizers can be reversed using association with targeting
biomacromolecules, such as protein.

6. Electron transfer mechanism and antimicrobial photodynamic
therapy
PDT can be applied for disinfection and sterilization [4–7]. Microbial, including
bacterium and viruses can be removed by photosensitized reaction. The physical treatment, such as PDT, is advantageous against antibiotic-resistant bacteria[91, 92]. PDT for microbial treatment is called as aPDT and/or PACT. Red light(relatively long wavelength visible light) is used for aPDT. Because 1O2 can be easily
produced by relatively small energy photons, it is considered as the important reactive species for aPDT process. Phenothiazine dyes, such as Methylene Blue is usedas the photosensitizer for aPDT [93], because Methylene Blue can absorb relatively
long-wavelength visible light and its ΦΔ value is relatively large [94]. However, theaPDT mechanism has not been well-understand. Biological environments are undera hypoxic condition [95], the mechanism mediated by 1O2 generation mechanism
may be restricted. Therefore, the electron transfer mechanism may play an important role in the aPDT mechanism.
6.1 Photosensitized DNA damage through electron transfer
DNA is a potentially important targeting biomacromolecules for PDT and aPDT
[1–3, 28]. In the cases of DNA damage, the generation of reactive oxygen species,
such as 1O2 (Type II mechanism), and the direct oxidation of nucleobases throughphotoinduced electron transfer (Type I mechanism) are important. In general, O2•−
formation and following H2O2 and/or •OH production (Type II mechanism, minor)
require relatively shorter wavelength radiation, such as ultraviolet ray [28, 32, 33].
Therefore, the contribution of the O2•− generation (Type II minor) mechanism isconsidered to be small in the aPDT mechanism. As mentioned above, photosensitized 1O2 generation is the important mechanism of aPDT. Guanine is the selective
target of 1O2, and every guanine is oxidized by 1O2 in a DNA sequence [28, 33].
Similar to the 1O2 generation mechanism, guanine is also damaged through electrontransfer selectively [28, 32, 33]. However, single guanines in double-stranded DNA
and guanine residue in single-stranded DNA are resistant to electron transfer
mechanism, in the contrary to the 1O2 mechanism [28, 33]. Since π-π interaction between consecutive guanines decrease the Eox of guanine, the consecutive
14

Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

guanines, such as GG and GGG, are selectively oxidized through electron transfer
mechanism [77–79]. Similar compounds are produced of guanine oxidation throughthe both mechanisms of 1O2 generation and electron transfer [72].
The mechanism of DNA damage photosensitized by Nile Blue (Figure 9) has
been studied as a potential photosensitizing reaction [96]. The reported value ofΦΔ by Nile Blue is very small (0.005) [66, 97]. Therefore, Nile Blue is an appropriate model to examine the oxygen-independent mechanism. Nile Blue bound to
DNA strand through an electrostatic interaction and the fluorescence lifetime wasdecreased, supporting the electron transfer quenching. Using 32P-5′-end-labeled
DNA fragments, DNA damaging mechanism of Nile Blue was examined andconsecutive guanine damage was observed. From the analysis of DNA damaging
pattern, the contribution of DNA damage through electron transfer mechanism wasestimated to be 72% (the contribution of 1O2 mechanism is 28%). The ΔG of electron transfer from guanine to the S1 state of Nile Blue is negative (−0.15 eV) [96],and this value is considered to become smaller in the case of consecutive guanine, as
mentioned above [77–79]. The estimated kET value is relatively large (1.0 × 1010 s−1).
These values supported the electron transfer-mediated DNA oxidation. The mechanism of DNA damage photosensitized by Nile Blue is shown in Figure 9. Relevantly,
rhodamine-6G, a fluorescence dye, induces the electron transfer-mediated oxidation of DNA [98] and folic acid [64] with photoirradiation. In general, fluorescencedyes hardly photosensitize 1O2 generation. On the other hand, photooxidative
activity through electron transfer depends on the redox potential of molecules.
These results suggest that the electron transfer-oxidation becomes important PDT
mechanism for non-1O2 generating dyes.
6.2 Photosensitized protein damage through electron transfer
Photosensitized protein damage by Methylene Blue and its analogues (Figure 10).
were studied [99]. Similar to the cases of phosphorus(V) porphyrin photosensitizers,
HSA was used as the targeting biomacromolecules. DNA binding through electrostaticforce of these cationic compounds are well-known [40, 71, 74, 96, 100]. However,
the interaction between these cationic dyes and HSA is small and a hydrophobic

Figure 9.
Structure of Nile Blue and the proposed mechanism of guanine decomposition through photoinduced electrontransfer.
15

Photodynamic Therapy - From Basic Science to Clinical Research

Figure 10.
Structures of Methylene Blue and its analogues. Binding constants with HSA were examined in a 10 mM
sodium phosphate buffer (pH 7.6). QET: The quantum yield of HSA oxidation through electron transfer
mechanism. QSO: The quantum yield of HSA oxidation through 1O2 generation.

interaction (not electrostatic interaction) may be a driving force of the association
with HSA [58]. The reported binding constant, which were estimated by the BenesiHildebrand Equation [101] are shown in Figure 10. Fluorometry of HSA tryptophan
residue demonstrated the photosensitized oxidation through both mechanisms,
electron transfer and 1O2 generation [99]. The analyzed quantum yields through thesemechanisms are shown in Figure 10. Fluorescence decay of these dyes was complex.
From the analysis of their observed fluorescence decay, the estimated kET values wereorder of 109 s−1, supporting the electron transfer mechanism. Furthermore, this result
suggests the existence of markedly fast electron transfer species, much faster than thedetection limit of this study (within ~50 ps) [99]. DFT calculation also supported theelectron transfer mechanism. The energy gap between the highest occupied molecularorbital (HOMO) of amino acids and that of photosensitizers are important for theelectron transfer mechanism. The plot between the HOMO values of these cationicdyes and the protein damaging quantum yield through electron transfer demonstrated
a relatively good relationship. Furthermore, the relationship between the ΦΔ and thedamaging quantum yield through 1O2 generation is also observed. These results shownthat the electron transfer mechanism is also important for photosensitized proteinoxidation by Methylene Blue and its analogues, as 1O2 generation mechanism does.
The electron transfer mechanism is not completely independent of oxygen molecule,because oxygen support the electron transfer by removing the excess electron fromthe reduced photosensitizer. However, other endogenous oxidative agents, such as
metal ions, may support the electron transfer mechanism, in vivo, the electron transfer mechanism may play an important role in the aPDT under hypoxic condition.

7. Conclusions
This chapter reviewed the several topics about the photosensitizers, which playelectron transfer-supported mechanism. 1O2 is the important reactive species in PDT
and aPDT. However, hypoxic condition in biological environment is not appropriate for reactive oxygen-dependent mechanism. Electron transfer is not completely
independent of oxygen; however, this mechanism does not absolutely require oxygen. Endogenous oxidative substances other than oxygen can support the electron
16Electron Transfer-Supported Photodynamic Therapy
DOI: http://dx.doi.org/10.5772/intechopen.94220

transfer mechanism. In the study of PDT photosensitizer for cancer, phosphorus(V)porphyrins showed the selectivity for cancer cell and relatively strong PDT effects.
Most important property of these photosensitizers is strong photooxidative activity through electron transfer under long-wavelength visible light irradiation.
Furthermore, the photosensitizing activity of phosphorus(V) porphyrins throughelectron transfer mechanism can be controlled by surroundings, such as pH. In
the processes of aPDT, the electron transfer mechanism may be important. For
developing the effective drugs for aPDT, molecular design based on the electrontransfer is also useful as well as that based on the 1O2 generating activity. The activityof electron transfer oxidation depends on the redox potential, and a long lifetimeof photoexcited state is advantageous. For PDT photosensitizers, relatively strong
response to long-wavelength radiation is required. In the molecular design of PDT
photosensitizers including phosphorus(V) porphyrins, the calculations of HOMO
energy level and the excitation energy are important as the initial steps.

Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific Research (B)from Japan Society for the Promotion of Science (JSPS KAKENHI 17H03086) andFutaba Electronics Memorial Foundation (10407).

Conflict of interest
The author declares no conflict of interest.

Author details
Kazutaka Hirakawa1,2
1 Department of Engineering, Applied Chemistry and Biochemical Engineering
Course, Graduate School of Integrated Science and Technology, Shizuoka
University, Hamamatsu, Japan
2 Department of Optoelectronics and Nanostructure Science, Graduate School of
Science and Technology, Shizuoka University, Hamamatsu, Japan*Address all correspondence to: hirakawa.kazutaka@shizuoka.ac.jp© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the termsof the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.
17

Photodynamic Therapy - From Basic Science to Clinical Research

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