Enhancement on the Hypocrellin B Singlet Oxygen
Generation Quantum Yield in the Presence of Rare Earth
Ions
Daniel José Toffoli*, Laércio Gomes*, Nilson Dias Vieira Junior*, Lilia Coronato
Courrol¶
*
Centro de Lasers e Aplicações, Instituto de Pesquisas Energéticas e Nucleares, IPEN–CNEN/SP, Sao Paulo, Brazil
¶
Universidade Federal de São Paulo – UNIFESP, Sao Paulo, Brazil
Abstract. This paper shows a study of the optical properties of the photosensitizer hypocrellin B and its complexes
formed with the rare earth ions lanthanum, europium and terbium, in ethanol and dimethyl sulfoxide (DMSO)
solutions, with the purpose of verify its potentiality for use in Photodynamic Therapy. Indeed, lanthanide ions are able
to modify hypocrellin B energy levels and radiative decay probabilities. Lifetime and infrared emission measurements
were employed in order to reveal the capacity of the complexes at generating singlet oxygen. Hypocrellin B complex
with lanthanum in ethanol showed the best results regarding ideal photosensitizers, since it displaced hypocrellin B
absorption peak at 584 nm to 614 nm, as well as enhanced singlet oxygen generation from 0.47 to 0.62 (reference:
methylene blue, Φ∆ = 0,52).
INTRODUCTION
Photosensitizers (PS) are chromophores with high molar absorptivity in the visible region and low toxicity to
biological tissues. When submitted to specific wavelength irradiation, they are able to induce or participate of
photochemical reactions with non-absorber molecules presents in the environment [1].
If this environment is rich in molecular oxygen, electron transfer or energy transfer reactions may occur (type I
and type II reactions, respectively), in which are generated reactive oxygen species (ROS) and singlet oxygen (1∆g).
Such substances are able to induce chain reactions with cell components, as well as oxidize a large variety of
biomolecules, owing to their high transmembrane potential. This way, ROS and 1∆g may cause necrosis (by direct
lethal effects or by vasculature damage) or apoptotic processes [2].
It is verified, hence, that solely in the presence of photons which wavelength is resonant with PS energy levels
and of molecular oxygen the PS becomes cytotoxic. This is the basic principle which supports the termed
Photodynamic Therapy (PDT), a technique known since many years ago but only widely studied in the last two
decades. PDT shows a great potential for treatment both of neoplastic injuries (selective ablation of tumoral tissue)
and non-neoplastic injuries (including ophthalmology, dermatology, cardiology, viral and bacterial inactivation and
blood purification) [3]. PDT stands out regarding other techniques (especially in cases of tumor treatment) due to its
great selectivity, which comes from two factors: the affinity among the PS and the target tissue and the illumination
restriction for the healthy tissues [1-3].
PDT mechanisms, I and II, previously cited, are permitted to occur only if the PS molecule, since excited to a
higher singlet state (Sn), undergoes non-radiative decays (internal conversions, ic), reach the first singlet excited
state (S1) and, through intersystem crossing (isc), have its spin changed and be led until the first triplet excited state
(T1). These processes are represented in the Jablonski diagram, as shown by Fig. 1.
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CP992, RIAO/OPTILAS 2007, edited by N. U. Wetter and J. Frejlich
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THE PAPER
ON
PP.or507
- 512
FIGURE 1. Jablonski diagram illustrating the various processes that may occur with the PS molecule after absorption of a
photon with resonant energy to one of its energy levels [4].
When the energy transfer between the PS’s first triplet excited state (T1) and oxygen in its ground state (3Σg-,
also a triplet state) takes place, formation of singlet oxygen (1∆g) is verified. 1∆g corresponds to the lowest excited
state of 3Σg- (it lies at only 94 kJ.mol-1 = 7900 cm-1 above the triplet ground state) [4,5]. Selection rules strictly
prohibit its luminescent decay for the ground state (which must involve multiplicity change), so the lifetime of the
isolated molecule is expected to be large. However, collisions among molecules of singlet oxygen may weak this
prohibition. Because of its singlet multiplicity no spin-prohibition exists for reactions with the most of organic
molecules, which are singlet in their ground state. Besides, the presence of an empty molecular orbital in 1∆g gives to
it electrophilic character. Thus, singlet oxygen is expected to be extremely reactive and to interact heavily with a
large number of biological substrates [1-5].
Both the pathways, type I and II, lead to oxidative stress, which gives rise to biological lesions in the target
tissue. As these are extremely reactive species, the diffusion coefficient of these photoproducts do not reach high
values, thus reinforcing the selectivity of the method. Obviously, complexity of biological systems affects the in vivo
answer of PDT. Specific enzymes may react with some ROS formed, this way minimizing the PDT effects;
nevertheless, 1∆g is not directly affected by many enzymes [6]. Therefore, the efficiency in PDT treatment is
attributed to a higher singlet oxygen generation quantum yield.
Innumerous PS, ranging from plant abstracts to complex synthetic macrocycles, were and have still been being
studied in order to turn PDT more efficient. In a general way, it is searched a chemically pure, known composition
compound which presents minimum toxicity in the dark. Moreover, it must be preferentially retained in the target
tissue – it was seen the tumor selectivity rises with the PS lipophilic character, which is in harmony with the fact that
neoplastic cells have a particularly large number of membrane receptors for low density lipoproteins, LDL (due to
their high demand for cholesterol) [7]. PS must have high pharmacokinetics (which would lead to low systemic
toxicity). It is also expected a large value for the PS T1 lifetime, which would permit sufficient time to energy
transfer reactions to occur, and high absorptivity coefficient in wavelengths ranging from 600 to 800 nm, region in
which light penetration in tissues reaches a maximum value (termed therapeutic window). At last, PDT efficiency
depends on the number of absorbed photons per volume tissue unity [1-4].
In the end of the 90’s, Photofrin® (a mixture of hematoporphyrin derivatives) was approved by FDA/USA for
clinical use in PDT of tumors as a photosensitizer agent. Subsequently, its use was also approved at Canada,
Germany, France, Brazil and Japan [1]. Unfortunately, some intrinsic problems of this drug, as prolonged
photosensibility and low 1∆g generation, incentive the research for new chromophores (or derivatives) with
improved physicochemical, pharmacokinetic and pharmacodynamic properties – the so called second generation of
PS [1,2]. Among such new PS stands out hypocrellin B (HB), Fig. 2, a perylenequinonoid pigment extracted from
Hypocrella bambusae fungus.
This compound presents some advantages as regards the hematoporphyrin derivatives, such as easy preparation
and purification, low aggregation tendency, high singlet oxygen generation quantum yield and fast in vivo
metabolism [8].
FIGURE 2. Molecular structure of hypocrellin B (C30H24O9).
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However, from the viewpoint of clinical applications, improvements in water solubility and in absorption
intensity at the 600-800 nm region are desired. Ma et. al. [8] found that complexes of hypocrellin B formed with
aluminum ions are able to fulfill these requirements, as well as to improve the generation of superoxide radical.
Nevertheless, singlet oxygen generation quantum yield is drastically reduced, which limits the applicability of this
complex. Zhou. et. al. [9,10] showed the complex of hypocrellin A (HA, a homologous pigment) formed with
lanthanum ion is responsible for similar enhancements in the water solubility and in the absorption band; however,
in spite of the reduction in the type II PDT mechanism noted in the complex of HB with aluminum as regards HB, a
improvement in the 1∆g generation is observed, as regards HA.
Thereby, the optical behavior of HB complexes formed with different rare earth ions (europium – Eu3+,
lanthanum – La3+ and terbium – Tb3+) was investigated with the objective of identify the composition which presents
the best optical and photochemical properties, i.e., the complex of hypocrellin B with rare earth ion which is the
most suitable for use in PDT.
MATERIALS AND METHODS
Hypocrellin B, methylene blue (MB), lanthanide chlorides (LaCl3.7H20, EuCl3.6H20 and TbCl3.6H20) and the
employed solvents, ethanol (CH3CH2OH) and dimethyl sulfoxide (DMSO, (CH3)2SO), were purchased from SigmaAldrich Corporation Ltd., with analytical grade (99% pure). It were prepared solutions of HB, HB:La3+, HB:Eu3+
and HB:Tb3+ in both solvents, following molar ratio 1:1 between HB and the lanthanide ions. It were obtained
concentrations of 1,89 mM for ethanol solutions and of 0,95 mM for those in DMSO.
Optical absorption was analyzed through Olis Cary 17-D spectrophotometer, from Varian, and a cuvette with
optical path of 1 mm, in order to verify the conformity of the PS absorption band with the area in which light
penetration in tissues reaches a maximum (600-800 nm). For emission experiments in visible, lock-in technique was
employed. The excitation source consisted of a xenon lamp (300 W) and a 0.25 m Jarrel-Ash monochromator.
Sample’s luminescence was guided to a 0.5 m Spex monochromator, being detected by a Hamamatsu
photomultiplier tube and amplified by an EG&G 7220 lock-in. In order to avoid any external noise, it was used a
Stanford chopper, put on the optical path of excitation and connected to lock-in. Optical path was 1 mm. By analysis
of emission and absorption spectra it was possible to determine the energies of the first singlet excited state (S1) and
of the first triplet excited state (T1) for all complexes. Infrared emission at about 1270 nm was verified with an
emission system from Edinburg Instruments. Such system was constituted by a Nd:YAG laser continuum Surelite
III, 5 ns/pulse, frequency of 10 Hz, a photomultiplier tube Hamamatsu R5509 and a cuvette with 1 cm optical path.
The emission intensities made possible determination of singlet oxygen generation quantum yield (Φ∆). Lifetime
measurements were obtained with a Opotek Nd:YAG+OPO laser excitation, a 0.25 m Kratos monochromator, S-20
photomultiplier tube, a 1300 nm filter and an oscilloscope. 1∆g lifetime (τ∆) and also Φ∆ were determined from these
measurements.
RESULTS AND DISCUSSION
In Fig. 3 are shown the optical absorption spectra for samples in ethanol and in DMSO. Firstly, one may note all
samples have a very large absorption band in the visible area, ranging from about 400 to 650 nm. These spectra also
confirm complexation of HB molecule by the rare earth ions, since changes in absorption peaks (then in energy
levels) are present in HB:La3+, HB:Eu3+ and HB:Tb3+.
1,75
1,50
Solvent: ethanol
HB
3+
HB:La
3+
HB:Tb
3+
HB:Eu
Absorbance (a. u.)
1,25
1,00
0,75
0,50
Solvent: DMSO
HB
3+
HB:La
3+
HB:Tb
3+
HB:Eu
(b)
1,25
Absorbance (a. u.)
(a)
1,50
1,00
0,75
0,50
0,25
0,25
0,00
350
400
450
500
550
600
0,00
350
650
Wavelength (nm)
400
450
500
550
600
650
Wavelength (nm)
FIGURE 3. Optical absorption spectra: (a) HB solutions in ethanol. (b) HB solutions in DMSO.
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TABLE 1. Absorption and luminescence peaks for all samples.
ETHANOL SOLUTIONS
DMSO SOLUTIONS
HB
HB:La3+
HB:Eu3+
HB:Tb3+
HB
HB:La3+
HB:Eu3+
HB:Tb3+
A
a
460
484
484
466
470
470
470
473
λ 1 (nm)
546
571
571
566
549
552
552
563
λA2 (nm) b
584
614
614
600
590
598
598
608
λA3 (nm) c
Inorm470 (a.u.) d
1.23E-3
0.12E-3
0.02E-3
0.36E-3
23.09
32.24
76.95
17.24
612
650
654
648
662.5
660
658.5
663
λΕmax (nm) e
a A
λ 1 = first absorption wavelength; bλA2 = second absorption wavelength; cλA3 = third absorption wavelength; dInorm470 =
maximum emission intensity normalized by absorbance in the excitation wavelength; eλΕmax = wavelength correspondent to the
maximum luminescence intensity.
Table 1 show these absorption peaks changes. From the viewpoint of clinical applications, HB:La3+ and
HB:Eu3+ in ethanol and HB:Tb3+ in DMSO showed the best results (remarkable enhancement in absorbance by a
factor of almost 102 and red-shift of 30 nm (ethanol samples) and 18 nm (DMSO samples)) thus leading HB closer
to an ideal PS.
In Fig. 4 are displayed samples emission spectra under excitation at 470 nm (wavelength in which all samples
have large absorption) and, also in Table 1, the wavelengths (λEmax) which correspond to maximum signal intensities
(Inorm470). One can see that all solutions show a large emission band, ranging from about 575 to 750 nm. Again the
question of complexation of HB molecule is elucidated, since it can be evidently seen modifications in radiative
decay probabilities before addition of rare earth ions in HB.
From both absorption and emission spectra the energy levels of the first singlet (S1) and the first triplet (T1)
states may be estimated. For each sample, absorption and emission spectra were plotted with the same maximum
value. The wavelength in which there is interception of the two curves represents, with good accuracy, the photon
emitted in the transition from S1 to S0 – so, S1 energy may be calculated from this point –, and the wavelength (in
nm) of the lower peak of the emission spectra represents T1. This way, Table 2 could be elaborated.
The excitation energy for singlet oxygen has a well know value, of 7900 cm-1 [4, 5]. Since for singlet oxygen
generation must occur energy transfer from the first triplet state of PS to the ground state of molecular oxygen,
compounds which T1 has a lower value than the excitation energy for molecular oxygen are generally not able to
generate 1∆g. As may be seen from Table 3, all samples have T1 > 7900 cm-1, which indicates their potentiality in
PDT.
0,0014
λexc = 470 nm
(a)
Solvent: ethanol
250
HB
3+
HB:La
3+
HB:Eu
3+
HB:Tb
200
Signal (a. u.)
0,0010
Signal (a. u.)
0,0012
0,0008
0,0006
λexc = 470 nm
(b)
Solvent: DMSO
HB
3+
HB:La
3+
HB:Eu
3+
HB:Tb
150
100
0,0004
50
0,0002
0
0,0000
550
575
600
625
650
675
700
725
750
550
575
600
625
650
675
700
725
750
Wavelength (nm)
Wavelenth (nm)
FIGURE 4. Luminescence spectra of samples (a) in ethanol and (b) in DMSO, under excitation at 470 nm.
-1
S1 (cm )
T1 (cm-1)
∆E (cm-1) a
a
∆E = S1 – T1
TABLE 2. Energies of the first excited singlet (S1) and triplet (T1) states.
ETHANOL SOLUTIONS
DMSO SOLUTIONS
HB
HB:La3+
HB:Eu3+ HB:Tb3+
HB
HB:La3+
HB:Eu3+
17,123.29 16,025.64 15,948.98 16,722.41 16,778.52 16,233.77 16,207.46
16,366.61 15,384.62 15,267.18 15,432.10 16,286.64 15,174.51 15,174.51
756.68
641.02
681.80
1,290.31
491.88
1,059.26
1,032.95
HB:Tb3+
15,673.98
15,082.96
591.02
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TABLE 3. Singlet oxygen lifetimes (τ∆) and singlet oxygen generation quantum yields (Φ∆) for ethanol and DMSO solutions.
ETHANOL SOLUTIONS
DMSO SOLUTIONS
3+
3+
3+
HB
HB:La
HB:Eu
HB:Tb
HB
HB:La3+ HB:Eu3+ HB:Tb3+
21.88
25.56
32.79
15.61
0.98
0.98
0.97
1.10
τ∆ (µs)
0.47
0.62
0.03
0.42
0.76 [8]
0.33
0.30
0.14
Φ∆
Singlet oxygen lifetimes (τ∆) were measured and are shown in Table 3. As may be seen, there is a large
difference between the values of τ∆ for samples in different solvents. It is known that singlet oxygen lifetime has a
strong dependence on the nature of the solvent [4]. Our measurements show that τ∆ in ethanol is much greater than
in DMSO, at about 20 times. A large value of τ∆ may imply a better treatment with PDT, since 1∆g would have
sufficient time to reach a wider area in the target tissue before lose energy by emission of photons.
In Fig. 5 (a) are presented the emission spectra of the samples in ethanol at infrared area of electromagnetic
spectrum, under excitation at 532 nm. MB was employed as reference (Φ∆ΜΒ = 0.52 [11]) for determination of the
singlet oxygen quantum yields displaced in Table 3 for the ethanol solutions. Indeed, emission at this wavelength is
due to the transition from singlet oxygen to ground state oxygen [4]:
1
(1)
∆g → 3Σg- + hν (1270 nm).
It is very important to emphasize that MB solution was made either in ethanol, as the samples analyzed through
this method. Finally, singlet oxygen luminescence efficiency, ηL, may vary with the environment. This way, it was
utilized the following equation in order to calculate the values of Φ∆:
Φ S∆ =
IS A R R
.
.Φ ∆
IR AS
(2)
where I is the signal intensity at 1270 nm, A, the absorbance in 532 nm (the excitation source) and the indexes S
means sample and R, reference.
It may be observed from Fig. 5 and Table 3 that only lanthanum addition at HB in ethanol is able to improve the
value of HB’s Φ∆ (from 0.47 to 0.62). Europium addition was responsible by quenching singlet oxygen generation,
while terbium addition did not induce remarkable changes in Φ∆.
Indeed, these results were expected, since in Fig. 4 (a) one may verify suppression on HB luminescence after
lanthanum addition. This fact leads to a higher value for the non-radiative decay rate constant, knr. It also indicates
there are other decay processes competing with luminescence, and one of them could be intersystem crossing –
which would lead to a better triplet state yield, ΦT. Furthermore, Table 2 shows diminution in the gap between S1
and T1 (∆E) for this complex when compared to pure HB, what could favor non-radiative transitions and also lead to
a higher ΦT value. This could explain the high value of Φ∆ for HB:La3+ in ethanol.
10000
Solvent: ethanol
HB
3+
HB:La
3+
HB:Eu
3+
HB:Tb
MB
(a)
3
Photon Counting (10 )
9000
7500
6000
(b)
Solvent: DMSO
HB
3+
HB:La
3+
HB:Eu
3+
HB:Tb
8000
Signal (a.u.)
10500
λexc = 532 nm
4500
6000
λexc = 532 nm
4000
3000
2000
1500
0
0
1230
1240
1250
1260
1270
1280
1290
1300
1310
1320
0,0
500,0n
1,0µ
1,5µ
2,0µ
2,5µ
3,0µ
3,5µ
4,0µ
Time (s)
Wavelength (nm)
FIGURE 5. (a) Infrared luminescence spectra of samples in ethanol under excitation at 532 nm. (b) Luminescence decays of
samples in DMSO under excitation at 532 nm.
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Analogous considerations could be done for europium complex; however, it suppresses Φ∆. So, one is lead to
believe that the probability of non-radiative decay through internal conversion (ic) is much higher than the
probability of the molecule to suffer an intersystem crossing (and thus generate 1∆g).
For terbium complex, there are suppression in luminescence and increase in ∆E. As Φ∆ diminished, one could
also suggest the luminescence quenching led to a higher non-radiative decay probability, through ic.
Figure 5 (b) introduces luminescence decays of samples in DMSO. Considerations about Φ∆ for these samples
were done through the signals obtained during lifetime experiments (emission filter at 1300 nm) and the values were
also arranged in Table 3. Calculations of Φ∆ (equation 2) for these solutions were done with HB DMSO as standard
(Φ∆HB D MSO = 0.76 [8]). In DMSO, all complexes decreased the value of Φ∆ referring to HB.
It was noted La3+ addition at HB in DMSO induced enhancement in luminescence (Fig. 4 (b)) as well as in the
energy gap between S1 and T1 (Table 2), and indeed there was a reduction in Φ∆. Similar reasoning may be done to
europium addition in HB DMSO.
For terbium complex, such reduction in Φ∆ was also expected, although less remarkable than for that caused by
lanthanum and europium addition, since the raise in the energy gap in this case has a lower value. Nevertheless, the
quenching in Φ∆ is larger, and one could suggest the energy transfer from PS to environment is more efficient than
from that to molecular oxygen.
CONCLUSIONS
Absorption, visible and infrared emission and luminescence decay spectroscopic methods were employed in this
work with the purpose of determine the best photosensitizer among the complexes studied. Singlet and triplet energy
levels were calculated. DMSO seems to reduce effectiveness of PDT, since its solutions showed a very short value
for τ∆. Absorption spectroscopy revealed that HB:La3+ in ethanol were responsible for a red-shift of 30 nm (from
584 to 614 nm) in the red absorption peak and introduced an enhancement in Φ∆ (from 0.47 to 0.62), as regards HB,
while the other complexes with rare earth ions in ethanol reduced Φ∆. Therefore, HB:La3+ complex in ethanol may
be considered a promising new generation PS agent.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. Frank Herbert Quina, from Instituto de Química da Universidade de São
Paulo, for his valuables considerations about the obtained spectra and by his attention and time dispended. Thanks
must be dispensed for Drs. Mauricio Baptista and Divinomar Severino, also from this institution, for the
measurements of Φ∆. Eventually, the authors thank CAPES – Coordenação de Pessoal de Ensino Superior, for the
grant.
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