2688
J. Phys. Chem. B 2007, 111, 2688-2696
Photodynamic Properties of Hypocrellin A, Complexes with Rare Earth Trivalent Ions: Role of the Excited State Energies of the Metal Ions Zhanghua Zeng,†,‡ Jiahong Zhou,† Yan Zhang,† Rui Qiao,†,‡ Shengqin Xia,† Jinrong Chen,† Xuesong Wang,*,† and Baowen Zhang*,† Lab of Organic Optoelectronic Functional Materials and Molecular Engineering, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100080, P. R. China, Graduate School of Chinese Academy of Sciences, Beijing 100049, P. R. China ReceiVed: October 26, 2006; In Final Form: January 15, 2007
Fifteen complexes of hypocrellin A (HA) with rare earth trivalent ions (except Pm3+) along with the complex of HA with Sc3+ were prepared, and their photodynamic activities, including absorption in the phototherapeutic window (600-900 nm); water-solubility; triplet lifetime; generation of reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion radical (O2-•), and hydroxyl radical (OH•); generation of semiquinone anion radical; and affinity to DNA, as well as photosensitized damage on calf thymus DNA (CT DNA), were compared in detail using the UV-visible spectrum, fluorescence spectrum, spin-trapping EPR technique, and laser photolysis technique. All complexes exhibit a red-shifted absorption spectrum, an increased absorbance above 600 nm, improved water solubility, and an enhanced affinity to CT DNA over the parent HA. For ions that possess low-energy excited states, including Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, and Yb3+, the corresponding complexes show undetectable or nearly undetectable fluorescence, a triplet excited-state lifetime, generation of ROS, and photodamage in CT DNA. In contrast, for ions that do not possess low-energy excited states, including Sc3+, Y3+, La3+, Gd3+, and Lu3+, the corresponding complexes exhibit higher photodamage abilities with CT DNA with respect to HA, benefitting from both their comparable or even higher 1O2 quantum yields and an electrostatic affinity that is higher for DNA than HA.
1. Introduction Photodynamic therapy (PDT), a promising treatment for cancer and for certain noncancerous conditions, utilizes reactive oxygen species (ROS), such as singlet oxygen (1O2), the superoxide anion radical (O2-•), and the hydroxyl radical, to attack the desired targets, whereas the generation of ROS requires a series of photophysical and photochemical processes of a photosensitizer induced by exposure to visible or nearinfrared light.1-4 The first PDT photosensitizer to receive regulatory approval in several countries was Photofrin. However, the limitations of Photofrin, particularly its long-standing skin photosensitivity, have encouraged the development of many new photosensitizers, including both porphyrin-like and non-porphyrin structures.3,5 As a new type of non-porphyrin photosensitizer, hypocrellin A (HA) and hypocrellin B (HB), naturally occurring perylenequinonoid pigments isolated from Hypocrella bambuase, have been well recognized to be efficient photodynamic agents against many tumor cell lines and viruses.6-10 Nevertheless, the poor aqueous solubility and the low absorbance in the phototherapeutic window (600-900 nm) hamper their clinical applications, which in turn has driven the synthesis and investigation of chemically modified hypocrellins.11-16 In this context, the metal complexes of hypocrellins are very attractive because they can be prepared with simple procedures and the enhanced water solubility and improved red absorptivity can be achieved * Corresponding authors. Tel: 86-01-82543592; Fax: 86-01-62554670; E-mails:
[email protected],
[email protected]. † Technical Institute of Physics and Chemistry. ‡ Graduate School of Chinese Academy of Sciences.
simultaneously.17,18 Recently, we prepared a complex of HA with lanthanum ion (La3+-HA), which to our surprise shows higher 1O2 quantum yield than the parent HA.19,20 In contrast, the 1O2 quantum yield of the complex between Al3+ and HA is much lower than that of HA.17 La3+-HA also exhibits longer triplet excited-state lifetime over HA and a larger electrostatic attraction to DNA and, thus, higher photodamage capability on calf thymus DNA (CT DNA).19,20 The remarkable and positive effects of La3+ on the photodynamic properties of HA aroused an interesting question, that is, how do the other lanthanide or rare earth ions act? In fact, some lanthanide complexes, for example, lutetium texaphyrin (Lutex or Motexafin Lutetium) and gadolinium texaphyrin, had been investigated systematically for applications in PDT and radiation therapy.3,21 The photophysical properties, including the 1O2 quantum yields, of texaphyrin complexes with lanthanide ions were also studied in-depth and correlated to their photodynamic activities.22 Several lanthanide texaphyrin complexes have been in clinical trials.3 In this work, the complexation of 15 rare earth trivalent ions (from Y3+ to Lu3+, not including Pm3+) with HA was investigated, and the ROS generation ability and DNA photodamage ability of the complexes were compared in detail. For comparison, the HA complex with Sc3+, the lightest metal ion in the IIIb group, was also studied. All complexes exhibit a red-shifted absorption spectrum, increased absorbance, improved water solubility, and enhanced affinity to CT DNA over the parent HA. It is observed that the paramagnetic ions behaved similarly to each other but significantly different from the diamagnetic ions, with the exception of Gd3+. The HA complexes of Sc3+, Y3+, La3+, Lu3+, and Gd3+ photodamaged
10.1021/jp067020t CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007
Photodynamic Properties of Hypocrellin CT DNA more efficiently than parent HA. In contrast, the photodynamic activity of the other paramagnetic ion complexes of HA decreased markedly. The underlying mechanism is discussed in terms of the excited-state levels of the metal ions. 2. Experimental Section 2.1. Chemicals. HA was isolated from fungus sacs of hypocrella bambusae and recrystallized three times from acetone before use. YCl3·6H2O, LaCl3·7H2O, CeCl3·7H2O, NdCl3·6H2O, EuCl3·6H2O, TbCl3·6H2O, DyCl3·6H2O, HoCl3·6H2O, YbCl3· 6H2O, and LuCl3·6H2O were purchased from Acros Organics. ScCl3·6H2O, PrCl3·6H2O, SmCl3·6H2O, GdCl3·6H2O, ErCl3· 6H2O, TmCl3·6H2O, 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), 2,2,6,6-tetramethyl-4-piperidone (TEMP), 9,10-diphenylanthracene (DPA), 1,4-diazabicyclo[2.2.2]octane (DABCO) and sodium azide (NaN3), were purchased from Aldrich. Ethidium bromide (EB), CT DNA, and superoxide dismutase (SOD) were purchased from Sigma. N,N-dimethylsulfoxide (DMSO) was dried over K2CO3 and distilled prior to use. Anhydrous ethanol and other chemicals of analytical grade were obtained from Beijing Chemical Plant. Water was freshly distilled twice before use. All experiments involving CT DNA were performed in PBS buffer solution (pH ) 7.4), unless otherwise noted. CT DNA solutions were prepared by dispersing the desired amount of DNA in buffer solution with stirring overnight at temperature below 4 °C. In the experiments in which titration with DNA was required, the DNA solution was sonicated at 0 °C for 10 min using a Brason probe ultrasonicator. This operation significantly reduced the viscosity of the DNA solutions and permitted more accurate and precise titration. The concentration of CT DNA was expressed as the concentration of nucleotide and was calculated using an average molecular weight of 338 for a nucleotide and an extinction coefficient of 6600 M-1 cm-1 at 260 nm.23 2.2. Preparation of the Metal Complexes of HA. All metal complexes of HA were prepared following the method of La3+HA (Pm3+-HA was not prepared due to the radioactivity of Pm-147),19,20 but the molar ratios between metal chloride and HA (r ) [MCl3·xH2O]/[HA]) were different. For the HA complexes with Sc3+, Y3+, La3+, Gd3+, and Lu3+, r was 1.0, whereas an r of 2.0 was adopted for the other ions. Taking the HA complex of Ce3+ (Ce3+-HA) as an example, the synthetic procedures are as follows. A 0.37-g portion of CeCl3·7H2O (1.1 mmol) dissolved in ethanol was mixed with the ethanol solution of HA (containing 0.27 g of HA, 0.5 mmol), and the mixture was stirred for 10 h at room temperature in the dark under N2 atmosphere. After filtration and removal of the solvent, the residue was dissolved in deionized water and dialyzed against dehydrated ethanol employing a spectrapor membrane with a molecular weight cutoff of >5000; thus, the low molecular weight ( 470 nm). The aliquots were removed at various times, and their fluorescence emissions in the range of 525-800 nm were measured by exciting at 510 nm. The percentage of the binding site remaining at a given time (t) was calculated from eq 2,
(
% binding site remaining ) 100 × 1 -
)
I0 - I t (2) I0 - Ibuffer
where I0, It, and Ibuffer denote the integrated fluorescence intensities before irradiation, after t min of irradiation, and of DNA-free buffer, respectively. 3. Results and Discussion 3.1. Complexation of HA with IIIb Metal Ions. Similar to La3+,19,20 Sc3+ and the examined rare earth trivalent ions can form a complex with HA very easily, and the complexation processes can be monitored clearly by UV-visible spectra. Taking Gd3+ as an example, the three absorption peaks of HA at 581, 542, and 463 nm shift bathochromically upon increasing the concentration of Gd3+ (Figure 1, Table 1). In addition, except for the middle peak, the other two bands exhibit enhanced absorbance. Thus, the resultant complex has better lightharvesting efficiency than parent HA in the phototherapeutic window (600-900 nm). During the entire titration process, one set of isobestic points was observed, indicating the presence of only one form of complex. The compositions of the complexes were determined by both molar ratio and continuous variation methods.26 For the molar
λab max (nm)
λflmax (nm)a
Φf a
581 618 620 629 623 625 619 621 622 625 626 628 623 625 621 628 623
603 630 641 637 ND ND ND ND ND 635 631 ND ND ND ND ND 629
1.00 0.48 0.22 0.13 ND ND ND ND ND 0.11 0.01 ND ND ND ND ND 0.11
ND means not detectable.
ratio method, a series of ethanol solutions containing different concentrations of metal ions and a constant concentration of HA (25 µM) were prepared, and the absorbance at a wavelength in the range of 615-630 nm (depending on the absorption maxima of different complexes) was plotted against the molar ratio of [metal ion]/[HA] (Figure 2). Two straight lines of different slopes can be derived from the plot, and the molar ratio where the two lines cross reflects the composition of the complex. In the continuous variation method, the total concentration of metal ion and HA was kept constant (50 µM), and the absorbance change (Y) at the complex maximum with respect to that of the HA solution (50 µM) at the same wavelength was plotted against the molar fractions of HA (Figure 3). Thus, Y reaches the maximum at the molar fraction of HA identical to that in the complex. Except for Pr3+, all metal ions examined fall into two categories: Sc3+, Y3+, La3+, Gd3+, and Lu3+ form 1:1 complexes with HA, whereas the other 10 lanthanide ions form 2:1 complexes. In the case of Pr3+-HA, a metal-to-HA ratio of 1.8 was determined. Figurse 2 and 3 show the results of Gd3+ and Eu3+ as the examples. The stability constants of the complexes that possesses higher 1O2 quantum yield were measured to be 1.13 × 106 M-1 (Sc3+-HA), 3.49 × 106 M-1 (Y3+-HA), 7.87 × 106 M-1 (La3+-HA), 1.79 × 106 M-1 (Gd3+-HA), and 5.82 × 106 M-1 (Lu3+-HA). Complexation also gives rise to significant changes in IR and 1H NMR spectra of HA. HA exhibits a sharp absorption around 1610 cm-1 attributable to the stretching vibration of the quinonoid carbonyl groups. In the complexes, this band shifts to a lower frequency, indicating the participation of the quinonoid carbonyl groups in coordination.17,27,28 Additionally, a broad absorption band around 450-750 cm-1 appearing in the complexes may be attributed to a metal-O bond.17,27 The 1H NMR data show that the signals of the two phenolic hydroxyl groups of HA disappear on formation of the complexes, suggesting their participation in coordination. It is also observed that the complexation with all paramagnetic lanthanide ions leads to low field shifts of the protons at 5 and 8 positions of HA to different extents, in good agreement with their capabilities as chemical shift agents.29,30 For example, the chemical shift of protons at 5 and 8 positions of HA moved from 6.43 to 6.82 ppm upon complexation with Gd3+. Owing to the use of a dialysis membrane (molecular weight cutoff of 5000) in the purification, the obtained M3+-HA complexes should possess polymer-like structures. Taking all
Photodynamic Properties of Hypocrellin
J. Phys. Chem. B, Vol. 111, No. 10, 2007 2691
Figure 2. Molar ratio plots for Gd3+-HA (a) and Eu3+-HA (b) in ethanol obtained by plotting the absorbance at 625 or 622 nm as a function of the molar ratio of Gd3+ or Eu3+ to HA ([HA] ) 25 µM).
Figure 3. Job’s plots for the Gd3+-HA (left) and Eu3+-HA (right) in ethanol obtained by plotting the absorbance difference (Y) at 625 or 622 nm with respect to neat HA solution at the same wavelength as a function of the mole fraction of HA ([HA] + [M3+] ) 50 µM).
SCHEME 1: Possible Structures for HA Complexes with the Metal Ions Examined
the abovementioned findings into consideration, two possible structures are proposed, as shown in Scheme 1. One uses HA as bridging ligands to construct 1:1 M3+-HA polymeric complexes (M3+ ) Sc3+, Y3+, La3+, Gd3+, or Lu3+), and the other uses both HA and Cl- as bridging ligands to build 2:1 M3+-HA polymeric complexes (M3+ ) Ce3+, Nd3+, Sm3+, Eu3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, or Yb3+). Both structures may coexist in Pr3+-HA and, thus, show a molar ratio of 1.8. H2O or ethanol or both may be present in the complexes to satisfy the large coordination number of these metal ions.18,31 The preparation of the complexes was carried out in ethanol; thus, we measured their stoichiometry also in ethanol, in which both HA and the corresponding metal salt have good solubility. Considering the high stability constants, it seems not likely that the molar ratio of M3+-HA in the complex will change upon transfer from ethanol to water. The aqueous solutions of the
complexes did not undergo changes in the UV-vis absorption spectrum upon standing at 40 °C overnight, which is indicative of the stabilities of the complexes in water. However, at present, we cannot define the number of ethanol and water molecules that are present in the complex to act as ancillary ligands. It is really possible that the ancillary ligands undergo changes in number and in type (between ethanol and water) with the change of solvent from ethanol to water. We performed elemental analyses on two complexes, Gd3+-HA and Eu3+-HA, as the examples of the complexes with 1:1 and 2:1 stoichiometry, respectively. Taking chloride as the counterion and ignoring any possible ancillary ligands (ethanol or water), the formula of Gd3+-HA and Eu3+-HA can be expressed as [(C30H24O10Gd)+Cl-]n and [(C30H24O10Eu2Cl2)2+(Cl-)2]n; thus, the calculated values are C, 48.88; H, 3.28; Cl, 4.81; and Gd, 21.33 for Gd3+-HA, and
2692 J. Phys. Chem. B, Vol. 111, No. 10, 2007 C, 36.39; H, 2.44; Cl, 14.32; and Eu, 30.69 for Eu3+-HA. The measured values are, C, 47.14 and H, 3.51 for Gd3+-HA, which matches [(C30H24O10Gd)+Cl-](1.5H2O), and C, 35.03 and H, 2.81forEu3+-HA,whichmatches[(C30H24O10Eu2Cl2)2+(Cl-)2](2.0H2O). The results seem to support the stoichiometry obtained spectroscopically. However, it should be borne in mind that the information from elemental analysis is very limited due to the lack of knowledge on the exact type and number of the ancillary ligands for these complexes. Furthermore, the inductively coupled plasma atomic emission spectrometry (ICP-AES) and precipitation titration of the Mohr method24 were applied to determine the content of Gd, Eu, and Cl. The measured contents of Gd (21.06), Eu (29.98) and Cl (4.77 in Gd3+-HA and 13.02 in Eu3+-HA) are all in good agreement with the stoichiometry of 1:1 for Gd3+-HA and 2:1 for Eu3+-HA. In addition to the red-shifted spectrum and the enhanced absorbance, these complexes also show remarkably improved water solubility over the parent HA. 3.2. Comparison of Photophysical Properties. All complexes display similar UV-visible absorption spectra, that is, three peaks in the visible region and one of them in the phototherapeutic window (see Gd3+-HA in Figure 1 as an example), and the absorption maxima of the lowest energy absorption bands are collected in Table 1. Also included in Table 1 are the fluorescence maxima and relative fluorescence quantum yields of these complexes (assuming the fluorescence quantum yield of HA is one unity). The fluorescence quantum yields decrease gradually in the order of HA > Sc3+-HA > Y3+-HA > La3+-HA ≈ Gd3+-HA ≈ Lu3+-HA, probably due to the heavy atom effect, which promotes intersystem crossing from singlet to triplet excited state.32 However, the nearly undetectable fluorescence of the other complexes suggests that an alternative quenching mechanism may be operative, which is very likely the results of the energy transfer from the singlet excited-state of HA to the low-energy excited states of lanthanide ions. Though energy transfer from the triplet excited state of the ligand to the lanthanide ions is a more general pathway, the energy transfer directly occurred from the ligand’s singlet excited-state was really vindicated recently in a Eu3+ complex by time-resolved spectrum techniques.33 The energytransfer quenching mechanism is also in line with the fact that the three complexes of La3+-HA, Gd3+-HA, and Lu3+-HA show similar fluorescence quantum yields due to the lack of low-energy excited states in La3+, Gd3+ and Lu3+ (the lowest excited state of Gd3+ locates ∼32 000 cm-1 above the ground state, far higher than the singlet excited-state energy of 18 000 cm-1 for HA).34,35 In the case of Eu3+-HA, electron transfer from the HA singlet excited state to Eu3+ may also make a contribution to the fluorescence quenching, considering the high tendency of Eu3+ to be reduced to Eu2+ (the one-electron reduction potential of Eu3+ is only -0.35 V vs NHE). 35. 3.3. Comparison on 1O2 Quantum Yields. The EPR spin trapping technique using 2,2,6,6-tetraethyl-4-piperridone (TEMP) as spin trapping agent was used to detect the formation of 1O2 and compare the relative quantum yields. Taking Gd3+-HA as an example, the irradiation of oxygen-saturated DMSO solution of Gd3+-HA (0.2 mM) and TEMP (50 mM) with a 532-nm laser led to a typical three-line EPR signal with the hyperfine coupling constants RN ) 16.0 G and g ) 2.0056 (Figure 4a), attributable to TEMPO, the adduct of 1O2 and TEMP.36 Control experiments confirmed that Gd3+-HA, oxygen, TEMP, and light are all essential for the signal generation (Figure 4d). When DABCO or NaN3, both typical 1O2 scavengers,37 was present,
Zeng et al.
Figure 4. (a) EPR spectrum obtained upon irradiation (at 532 nm) of an O2-saturated DMSO solution containing 50 mM TEMP and 0.2 mM Gd3+-HA. (b) Similar to (a) but in DMSO-d6 solution. (c) Similar to (a) but in the presence of NaN3 (10 mM). (d) Similar to (a) but in the absence of Gd3+-HA, oxygen, TEMP, or light.
TABLE 2: Relative Quantum Yields of 1O2, O2-•, and Semiquinone Anion Radical for HA and Its Complexes in DMSO Solutions
photosensitizer HA Sc3+-HA Y3+-HA La3+-HA Ce3+-HA Pr3+-HA Nd3+-HA Sm3+-HA Eu3+-HA Gd3+-HA Tb3+-HA Dy3+-HA Ho3+-HA Er3+-HA Tm3+-HA Yb3+-HA Lu3+-HA a
1O 2
O2-• a
semiquinone anion radical a
1.00 0.28 0.50 1.32
