J. Phys. Chem. B 2005, 109, 19529-19535
19529
Effect of Chelation to Lanthanum Ions on the Photodynamic Properties of Hypocrellin A Jiahong Zhou,*,† Jihua Liu,§ Shengqin Xia,‡ Xuesong Wang,*,‡ and Baowen Zhang*,‡ Analysis & Detecting Central, Nanjing Normal UniVersity, Nanjing, 210097, People’s Republic of China, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China, and Department of Complex Presection of CMM, China Pharmaceutical UniVersity, Nanjing, 210038, People’s Republic of China ReceiVed: May 1, 2005; In Final Form: August 11, 2005
Hypocrellin A (HA), an efficient phototherapeutic agent, can chelate with lanthanum ion to form a 1:1 complex, which exhibits enhanced 1O2 generation quantum yield, a red-shifted absorption window, greatly improved water solubility, a much lengthened triplet excited state lifetime, and an increased affinity to DNA with respect to HA. These effects in turn lead to a more potent photodamage ability on calf thymus DNA for La3+-HA than HA in both aerobic and anaerobic conditions, indicating the potential application of La3+HA in the field of photodynamic therapy (PDT).
1. Introduction Hypocrellins including hypocrellin A (HA) and hypocrellin B (HB), isolated from the fungus of Hypocrella bambusae, have been receiving intensive interest over the past two decades in photodynamic therapy (PDT) due to their wide absorption band in the visible region and extremely high singlet oxygen (1O2) generation ability.1 From the viewpoint of clinical applications, the water solubility and absorption intensity in the phototherapeutic window (600-900 nm) of the natural hypocrellins need to be improved for attaining ideal photodynamic efficacy. Therefore, how to improve the water solubility and red absorption of hypocrellins has become the focus in the study of hypocrellins. Structural modification (such as sulfonated HA,2 glycosylated HB,3 cyclodextrin-modified HB,4 tyrosine-modified HB5) and complexing with metal ions6 are two approaches adopted to solve these problems. Comparing the two methods, the metal chelation complexes of hypocrellins are much easier to prepare. Moreover, the metal complexes of hypocrellins usually exhibit remarkable red shifts in absorption and greatly enhanced water solubility with respect to the natural hypocrellins. However, the chelation of hypocrellins to the metal ions generally results in a significant reduction of the 1O2 generation quantum yield,6 which unfortunately offsets the improvement in water solubility and light absorption. We recently communicated the high 1O2 generation quantum yield of a HA complex with lanthanum ion (La3+).7 In this work the influence of chelation upon La3+ on the abilities of HA to generate reactive oxygen species including superoxide anion radical (O2•-), hydroxyl radical (OH•), and 1O2 were investigated in detail using electron paramagnetic resonance (EPR) and spectrophotometric methods. It was found that the chelation of HA to La3+ (1) improves 1O2 generation, but reduces O2•- and OH• formation, (2) increases the triplet excited state lifetime, and (3) strengthens the affinity to DNA. The later two influences favor photodynamic damage on DNA by way of type I * Corresponding authors. Telephone: 86-25-8399-7183. Fax: 86-258359-8359. E-mail:
[email protected] (J.Z.). † Nanjing Normal University. ‡ Chinese Academy of Sciences. § China Pharmaceutical University.
mechanism under anaerobic conditions, and the improved 1O2 generation quantum yield favors photodynamic processes of type II mechanism under aerobic conditions. As a result, La3+-HA exhibited much higher photodynamic activities than HA under both aerobic and anaerobic conditions. 2. Materials and Methods 2.1. Materials. HA was isolated from the fungus sacs of Hypocrella bambusae and recrystallized twice from acetone before use. 2,2,6,6-Tetraethyl-4-piperidone (TEMP), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 1,4-diazabicyclo[2,2,2]octane (DABCO), LaCl3‚7H2O, superoxide dismutase (SOD), ethidium bromide (EB), and 9,10-diphenylanthracene (9,10DPA) were all purchased from Aldrich Chemical Co., USA. Dimethyl sulfoxide (DMSO), cysteine, N-methylaniline, sodium benzoate, anhydrous ethanol, hematoporphyrin, and other reagents of analytical grade were obtained from Beijing Chemical Plant. Water was freshly distilled before use. 2.2. Preparation of the Complex. LaCl3 (1 mM) and HA (1 mM) were mixed and stirred in ethanol for 10 min at room temperature in the dark. After completion of chelation, the mixture was filtered and the solvent evaporated to dryness from the filtrate under high vacuum. The residue obtained was redissolved in deionized water and dialyzed against dehydrated ethanol using a spectrapor membrane with a molecular weight cutoff of >3500, so the low molecular weight components ( 470 nm). The aliquots were removed at various times and analyzed with fluorescence emission spectra measured from 525 to 800 nm and excited at 510 nm. The percentage of binding sites remaining at a given time (t) was calculated from eq 2:
(
% binding sites remaining ) 100 1 -
I0 - I t I0 - Ibuffer
)
(2)
where I0, It, and Ibuffer denote the integrated fluorescence intensities before irradiation, after t min of irradiation, and of DNA-free buffer, respectively. 2.9. Chemiluminescence Assays for CT DNA Cleavage. DNA damage induced by photosensitizers was also assayed by the chemiluminescence method.10 The reaction mixture contained 25 µL of CT DNA (0.1 mg/mL), 25 µL of photosensitizers (HA or La3+-HA, 25 µmol/L phosphate buffer was used in the control), and 450 µL of 0.05 mol/L PBS (pH 6.5). Luminescence was counted for 60 s on a BPCL Model Ultra Weak Chemiluminescence Analyzer (Institute of Biophysics, Academia Sinica, Beijing, China) at 16 °C; the detector sensitivity was calibrated each time with a 14C light source at 10 000/s. All samples were measured immediately after 5 min of irradiation. 3. Results and Discussion 3.1. Formation of La3+-HA Complex. HA in ethanol solution has three absorption peaks at 581, 542, and 463 nm, respectively, in its visible absorption spectrum. When lanthanum ion was added to the ethanol solution of HA, the three absorption peaks red shifted gradually and the absorption intensity increased. At last, the positions of the three absorption peaks fixed at 627, 580, and 493 nm, respectively (Figure 1). This remarkable change in visible absorption spectra indicated that lanthanum ion could chelate with HA intensely. The composition of the complex was determined by molar ratio and continuous variation methods.11 For the molar ratio method, a series of ethanol solutions were prepared in which the concentration of La3+ was held constant (10 µM), while that of the HA was
Effect of Chelation to La Ions on HA Photodynamics
J. Phys. Chem. B, Vol. 109, No. 41, 2005 19531
Figure 3. Absorption spectrum changes of deoxygenated DMSO solution containing La3+-HA (85 µM) and cysteine (100 µM) upon irradiation for 0, 1, 2, 3, 4, and 5 min. The arrows indicate the direction of changes.
CHART 1: Schematic Representation of the Polymeric Complex of HA with La3+ Ion
Figure 2. (a) Mole ratio plot for La3+-HA complex in ethanol obtained by plotting absorbance at 630 nm as a function of mole ratio of HA to La3+. (b) Job plot for La3+-HA complex in ethanol. The vertical coordinate (Y) represents the difference between the absorbance (at 630 nm) of the mixed solution and that of the neat HA solution.
varied. The formation of the La3+-HA complex was detected by measuring the absorbance increase at 630 nm, where both La3+ and HA exhibit low absorption. As shown in Figure 2a, two straight lines of different slopes were observed, and the extrapolated intersection occurred at the mole ratio of 1:1, corresponding to the ratio of La3+ to HA in the complex. The association constant was calculated to be 8.26 × 106 M-1. For the continuous variation method, the total concentration of La3+ and HA was kept constant (50 µM), while the molar fractions of La3+ in the mixed solutions were continuously varied. The absorbance of each mixed solution was measured at 630 nm. The absorbance differences (Y) between the mixed solutions and the neat HA solution were plotted against the molar fraction of La3+. As shown in Figure 2b, the maximum Y occurred at the molar fraction of 0.5, supporting the 1:1 molar ratio of La3+ and HA in the complex, too. The IR spectrum of the complex shows that HA has chelated with lanthanum ion. The characteristic absorption band of the quinonoid carbonyl group in HA (1602 cm-1) shifted bathochromically to 1491 cm-1 on coordination of the carbonyl oxygen with La3+. In addition, the broad absorption band in the range 480-800 cm-1 also attests to the presence of La3+-O bond.6 The other evidence of chelation between HA and La3+ is from the significant change of water solubility. HA is insoluble in water; however, it can be easily dissolved in water upon chelation with La3+. As mentioned above, the purification of La3+-HA needed membrane tubing with a molecular weight cutoff of 3500, which allowed the small
molecular weight species to be removed, indicating that La3+HA might be a polymeric structure (Chart 1), and similar to that of Al3+-HA.6 The improved absorption in the phototherapeutic window and the enhanced water solubility with respect to HA endow La3+HA potential application in PDT. 3.2. Semiquinone Anion Radical of La3+-HA. When a nitrogen-saturated DMSO solution of La3+-HA (85 µM) and cysteine (used as electron donor, 100 µM) was irradiated with the visible light from a medium-pressure sodium lamp, all the absorption peaks of La3+-HA decreased, but the peak at 492 nm red shifted slightly, accompanied by three isosbestic points at 414, 515, and 644 nm within the spectral region examined (350-800 nm) (Figure 3). The absorption changes of the solution induced by irradiation can be observed easily by the naked eye. The color of the solution changed from green to yellow upon irradiation. If oxygen was allowed into the illuminated solution, the absorption spectrum of the La3+-HA was partly recovered. The observation mentioned above can be attributed to the photoinduced electron transfer between La3+HA and cysteine and the formation of the semiquinone anion radical of La3+-HA. The oxidation potential of cysteine is measured to be 0.70 V and the reduction potential of La3+HA is -0.43 V versus NHE, so the electron transfer from ground state cysteine to excited state La3+-HA is a thermodynamically favorable process (∆G ) -0.78 or -0.71 eV for excited singlet state or triplet state reaction, respectively), estimated by the Rehm-Weller equation (eq 3), where the singlet excited state energy of La3+-HA is calculated from the onset of the lowest energy absorption band (1.91 eV), and the triplet excited state energy of La3+-HA is assumed to be similar to that of HA (1.84 eV).12
19532 J. Phys. Chem. B, Vol. 109, No. 41, 2005
Zhou et al.
∆G ) Eox(donor) Ered(acceptor) - E0,0(excited state energy) (3) As a result, photoinduced electron transfer between La3+HA and cysteine gave rise to the formation of the semiquinone anion radical of La3+-HA, leading to absorption changes (eq 4). The presence of O2 in solution can oxidize the semiquinone anion radical of La3+-HA to La3+-HA, which led to the recovery of the absorption of La3+-HA (eq 5).
La3+-HA* + cysteine f La3+-HA•- + cysteine•+ La3+-HA•- + O2 f La3+-HA + O2•-
(4) (5)
We cannot rule out the involvement of hydroquinone in the photoinduced absorption changes. Hydroquinone may be generated by the disproportionation of the neutral semiquinone radical,13,14 which in turn results from the protonation of the semiquinone radical anion with either cysteine or cysteine cation or even trace water as the source of protons. It was reported that in acidic aqueous solutions the formed hydroquinone did not response to the introduced O2,13,14 which suggests that hydroquinone might be present in our irradiated solution, because O2 cannot give rise to a full recovery of the absorption spectrum in our cases. EPR technique was utilized to study the semiquinone anion radical of La3+-HA further. Irradiating the nitrogen-saturated DMSO solution of La3+-HA (50 µM) for 1 min at 532 nm, an ESR signal (Figure 4b) with the same position and line shape as that of the radical anion of HA was observed (Figure 4a),4b,15 but the signal intensity was much lower than that obtained with HA. The formation of radical anions of hypocrellins is believed to be the result of self-electron transfer between excited and ground state hypocrellins. Both free energy changes involved in the self-electron transfer of HA and La3+-HA are calculated to be -0.04 eV, using oxidation potentials of 1.66 V for HA16 and 1.44 V for La3+-HA, reduction potentials of -0.37 V for HA16 and -0.43 V for La3+-HA (all potentials are vs NHE), and singlet excited state energies of 2.07 eV for HA and 1.91 eV for La3+-HA. Though the calculated driving forces are the same (-0.04 eV), the much weaker semiquinone signal indicates the self-electron transfer is restrained in La3+-HA (eq 6), which suggests that the self-electron-transfer process may be diffusion controlled and therefore less efficient for La3+-HA due to its polymeric character.
La3+-HA* + La3+-HA f La3+-HA•- + La3+-HA•+ (6) When N-methylaniline was added to the deoxygenated DMSO solution of La3+-HA, the intensity of the EPR signal of the semiquinone anion radical of La3+-HA was enhanced significantly (Figure 4c), because N-methylaniline is a strong electron donor and can donate an electron to excited state La3+-HA to form the semiquinone anion radical of La3+-HA efficiently, similar to the case of cysteine. Bubbling air through the irradiated La3+-HA solution can make the EPR signal disappear immediately (Figure 4d), suggesting that the oxidation of La3+HA•- by O2 (eq 5) occurred. The EPR and spectrophotometric results showed that excited La3+-HA has the ability to undergo one-electron reduction to generate the semiquinone anion radical, which is essential for the generation of reactive oxygen species such as O2•- and OH• (see below).
Figure 4. (a) EPR spectrum from a deoxygenated DMSO solution of HA (50 µM) upon illumination at 532 nm for 1 min. (b) Similar to (a) but La3+-HA instead of HA. (c) Similar to (b) but in the presence of N-methylaniline (20 µM). (d) Similar to (b) but oxygen was allowed into the solution. (e) Similar to (b) but La3+-HA or light was omitted.
Figure 5. (a) EPR spectrum from an air-saturated DMSO solution of La3+-HA (100 µM) and TEMP (50 mM) upon illumination at 532 nm for 1 min. (b) Similar to (a) but in the presence of DABCO (50 mM). (c) Similar to (a) but in the absence of La3+-HA, oxygen, or light.
3.3. Singlet Oxygen Generation. It has been previously reported that 2,2,6,6-tetraethyl-4-piperidone-N-oxyl radical (TEMPO), a nitroxide radical detectable by EPR, was generated from the reaction of TEMP with singlet oxygen (1O2). When the oxygen-saturated DMSO solution of La3+-HA (0.1 mM) was irradiated at 532 nm for 1 min, and TEMP (10 mM) was used as a spin trap,17 an ESR spectrum of triplet peaks with equal intensity, characteristic of the signal of TEMPO, was observed (Figure 5a). The hyperfine splitting constant of the signal was 16.0 G, in line with the reported value for TEMPO.18 Under similar conditions but in the absence of La3+-HA or oxygen or irradiation, no ESR signals were observed (Figure 5c). To provide further evidence to support the involvement of 1O in this photosensitizing process, DABCO, a specific 1O 2 2 scavenger, was added to the solution of La3+-HA. After irradiation for 1 min, the ESR signal of TEMPO was also observed, but the intensity was suppressed (Figure 5b). These phenomena suggest that TEMPO is derived from the reaction of TEMP with 1O2 generated via the energy transfer from the triplet excited state of La3+-HA to ground state oxygen (eq 7).
(La3+-HA)* + O2 f La3+-HA + 1O2
3
(7)
1O is believed to be one of the reactive intermediates in PDT; 2 consequently, the quantum yield of 1O2 is an important parameter for the evaluation and optimization of the photosensitizers used in PDT. The photooxidation of 9,10-DPA to its endoperoxide derivative by singlet oxygen was used to determine the quantum yield of 1O2 (9,10-DPA bleaching method), taking hematoporphyrin as a reference. Figure 6 shows the rates of 9,10-DPA bleaching at 436 nm photosensitized by La3+HA, HA, and hematoporphyrin in DMSO solutions as a function
Effect of Chelation to La Ions on HA Photodynamics
Figure 6. (1) Photosensitized DPA bleaching by measuring absorbance decrease (∆A) at 374 nm as a function of irradiation time in oxygensaturated DMSO solution containing hematoporphyrin and DPA (50 µM). (2) Same as (1) except that hematoporphyrin was replaced by La3+-HA. (3) Same as (1) except that hematoporphyrin was replaced by HA. (4) Same as (1) except that hematoporphyrin (La3+-HA, HA), oxygen, or irradiation was omitted. Inset: Absorption spectra in the DPA bleaching system upon irradiation. The arrow indicates the direction of changes.
Figure 7. Transient absorption spectra of La3+-HA in deaerated DMSO solution at 200 ns, 10 µs, and 40 µs after the laser flash (532 nm). Arrows indicate direction of changes.
of irradiation time. Control experiments indicated that no 9,10DPA bleaching occurred when La3+-HA (or HA, or hematoporphyrin), oxygen, or irradiation was omitted (Figure 6, line 4). The 1O2 generation quantum yield of La3+-HA was calculated to be 0.90, and that of HA is 0.62, assuming that the 1O generation quantum yield of hematoporphyrin was unity. 2 Generally, metal-HA complexes, such as Al3+-HA,6 possess reduced 1O2 generation quantum yields with respect to HA. The improved 1O2 generation ability, in combination with the large absorbance above 600 nm and great water solubility, suggests La3+-HA is a promising candidate as a PDT sensitizer. 3.4. Time-Resolved Absorption Spectra. The generation efficiency of 1O2 depends on the photophysical properties of sensitizers, including the intersystem-crossing quantum yield, the triplet state lifetime, and the triplet state energy level. The time-resolved absorption spectra were measured to study the triplet excited states of hypocrellins.19 Figure 7 shows the timeresolved absorption spectra of a deaerated solution of La3+HA (50 µM) in DMSO after 532 nm laser excitation. There are three positive absorption bands with maxima around 578, 654, and 723 nm, respectively. The absorption below 530 nm is negative due to the depletion of the ground state La3+-HA.
J. Phys. Chem. B, Vol. 109, No. 41, 2005 19533
Figure 8. Dependence of ESR signal intensity of DMPO-superoxide radical adduct on irradiation time for an air-saturated DMSO solution containing La3+-HA (100 µM) or HA (100 µM) and DMPO (50 mM) irradiated at 532 nm. Inset: ESR spectrum of DMPO-superoxide radical adduct produced in irradiated solution.
The three positive bands can be attributed to the triplet-triplet (T-T) absorption of La3+-HA based on their efficient quenching by O2, which is similar to the reported T-T absorption of HA.19 The triplet excited state decay of La3+-HA, monitored at 570 nm (Figure 1S, Supporting Information), decayed monoexponentially with the lifetime of 179 µs, while the triplet lifetime of HA was 13 µs.20 The greatly lengthened triplet lifetime, which may be the result of the enhanced rigidity upon coordination and therefore the restricted nonradiative relaxation, is probably responsible for the increased 1O2 generation efficiency La3+-HA compared to that of HA. It has been reported that hypocrellins in their triplet multiplicity can interact with biomacromolecules directly in anaerobic conditions21 and give rise to the photodamage to biomacromolecules. In this regard, such a long-lived triplet state for La3+-HA is also beneficial to the phototherapeutic process carried out in oxygen-deficient cases. 3.5. Superoxide Anion Radical Generation. DMPO spin trapping has been successfully applied to detect certain reactive intermediates, in particular O2•- and hydroxyl radical, because it has a high affinity for these reactive radicals and leads to the formation of persistent spin adducts. When an air-saturated DMSO solution of La3+-HA and DMPO was irradiated with 532 nm light, a new EPR spectrum appeared (Figure 8, inset). This EPR spectrum can be characterized by three hyperfine coupling constants: RN ) 13.0 G, RβH ) 10.1 G, and RγH ) 1.5 G.22 It was due to the nitrogen and two hydrogen atoms at the β- and γ-positions, and was consistent with previously reported values for DMPO-O2•- adduct. Again, this signal could not be detected in the dark, or upon irradiation but omitting any of the sample components. The DMPO-O2•signal intensity generated in La3+-HA solution was lower than that in HA solution (Figure 8), consistent with the results in Figure 4, because O2•- was generated via electron transfer from semiquinone anion radical of La3+-HA to oxygen (eq 5). 3.6. OH• Generation. If water was present in air-saturated DMSO solution of La3+-HA and DMPO, irradiation with the 532 nm laser gave an ESR spectrum of the spin adduct DMPOOH• (Figure 9a), which was characterized by two coupling constants associated with the nonzero nuclear spins of the nitrogen atom and the hydrogen atom in the β-position: RN ) RH ) 14.9 G. The two identical coupling constants from β-N and β-H, which are in good agreement with the literature,23 give a four-line ESR spectrum with the intensity ratio of 1:2:2:1. The presence of sodium benzoate, a scavenger of hydroxyl radical, decreased the ESR signal significantly (Figure 9b).
19534 J. Phys. Chem. B, Vol. 109, No. 41, 2005
Zhou et al. TABLE 1: Photocleavage of CT DNA by La3+-HA or HA Detected by Percentage of Remaining Binding Sites (%) of Ethidium Bromide to Damaged CT DNA under Different Conditionsa irradiation time/min
Figure 9. (a) ESR spectrum of DMPO-Hydroxyl radical adduct resulting from irradiation of air-saturated buffer solution of La3+-HA (100 µM) and DMPO (50 mM). (b) Similar to (a), but in the presence of sodium benzoate (10 mM). (c) Removal of La3+-HA, oxygen, or light.
Control experiments ensured that no signal was obtained without light, oxygen, La3+-HA, or DMPO (Figure 9c). These observations confirmed the assignment of the signal to the DMPOOH• radical adduct. In addition, it was found that La3+-HA generates OH• with lower efficiency than HA does; the trend is similar to that of O2•- generation efficiency for La3+-HA and HA. Generally, there are two pathways to generate hydroxyl radical in hypocrellin photosensitization systems.24 One is via the superoxide-driven Fenton reaction (eqs 8 and 9), in which H2O2 originates from the dismutation of superoxide anion radical (eq 10) while Fe3+, Fe2+, or other transition metal ions with multiple oxidation states are often present in trace amount in aqueous solution.
Fe3+ + O2•- f Fe2+ + O2
(8)
Fe2+ + H2O2 f Fe3+ + OH• + OH•
(9)
O2•- + O2•- f H2O2 + O2
(10)
The other is via the reaction of semiquinone radical anion with H2O2 (eq 11):
La3+-HA•- + H2O2 f La3+-HA + OH• + OH•
(11)
Both pathways involve the generation of superoxide anion radical; this is why La3+-HA produces OH• less efficiently than HA. The generation of O2•-, OH•, and 1O2 by excitation of La3+HA indicates that (1) La3+-HA maintains the photodynamic activity in terms of type I and type II mechanisms and (2) photodynamic behavior of type II mechanism may be favored for La3+-HA under aerobic conditions due to the improved 1O generation quantum yield with respect to HA. 2 3.7. Affinity to CT DNA. Considering the positively charged polymeric structure of La3+-HA, one may expect that La3+HA can interact with the negatively charged double-strand DNA through electrostatic action, favoring the photodynamic damage onto DNA. For better understanding of the PDT behavior of La3+-HA, the affinity of La3+-HA to CT DNA was investigated by measuring its influence on the melting temperature (Tm) of CT DNA. Under our experimental conditions, the Tm of CT DNA (40 µM) in 10 mM sodium phosphate buffer (pH 7.4) is 60.1 °C. When adding 10 µM HA and La3+-HA to the CT DNA solution, respectively, the Tm of CT DNA decreased to 59.2 °C in the case of HA, but increased to 80.3 °C in the case of La3+HA. According to our previous work,25 HB, the analogue of
sample
10
20
30
40
50
control exptb La3+-HA + N2 HA + N2 La3+-HA + O2 HA + O2
99.92 96.81 99.62 93.59 95.63
99.78 93.01 98.29 86.17 92.02
99.65 90.62 95.55 78.65 87.72
99.45 87.10 92.57 72.00 84.66
99.28 82.42 90.07 66.30 79.09
[CT DNA] ) 40 µM, [EB] ) 80 µM, [La3+-HA] ) 5.6 µM, [HA] ) 5.6 µM. b In the absence of La3+-HA or HA, and oxygen. a
HA, can interact with CT DNA partly by intercalation mode, which increases the distance between the neighboring base pairs and makes CT DNA easier to be detached to single strand. In contrast, the surface binding of the positively charged polymeric La3+-HA may make CT DNA more difficult to be detached by virtue of the electrostatic interaction between them. It is clear that the affinity of La3+-HA to CT DNA is much larger than that of HA, leading to a large Tm change of 20.2 °C. ESR experiments also corroborate the improved affinity between La3+-HA and CT DNA. Irradiation of nitrogensaturated buffer solution of HA or La3+-HA with 532 nm laser can produce signals attributable to the semiquinone anion radical of HA or La3+-HA (Figure 2S). In the presence of the same amount of CT DNA, the semiquinone anion radical signals were intensified 1.4-fold in HA solution and 6.4-fold in La3+-HA solution, respectively. The increased signal intensity can be ascribed to the electron-donating property of CT DNA, of which the bases, particularly guanine, may efficiently donate an electron to the triplet excited state of HA or La3+-HA. The more significant ESR signal intensity enhancement effect occurring in the case of La3+-HA may arise from either the association of La3+-HA with CT DNA favored by electrostatic interaction between them or the longer triplet state lifetime of La3+-HA (80 µs in aqueous buffer solution) with respect to that of HA (0.1 µs in aqueous buffer solution). To clarify which factor is the predominant one, a neutral electron donor, diethylamine, was used to replace CT DNA in ESR experiments. Upon addition of the same amounts of diethylamine, the semiquinone anion radical signal intensities of HA and La3+HA were increased 3.8-fold and 2.7-fold, respectively. This indicates that the electrostatic interaction indeed has an important effect in the surface binding of La3+-HA to CT DNA, which in turn improves the affinity between La3+-HA and CT DNA and promotes the formation of semiquinone anion radicals via the electron transfer between triplet La3+-HA and CT DNA. 3.8. Photoinduced Damage to CT DNA. To study the PDT properties of La3+-HA, CT DNA in air-saturated buffer solution (10 mM acetic acid ammonium salt, 100 mM sodium chloride, pH 7.0) was used as phototherapeutic target and ethidium bromide (EB) assay was adopted to follow the photodamage process of CT DNA. Calculating from eq 2, 33.70% binding sites were destroyed during a 50 min irradiation with light above 463 nm in EB-CT DNA buffer solution containing 5.6 µM La3+-HA, while only 20.91% binding sites were damaged with the same concentration of HA as sensitizer under aerobic conditions (Table 1). Many cancerous cells grow in anaerobic circumstance; therefore, the photosensitized cleavage and damage to CT DNA by La3+-HA and HA were also investigated in the absence of oxygen. In such cases, the photodamage capability of La3+HA (17.58% binding sites destroyed) is almost twice that of
Effect of Chelation to La Ions on HA Photodynamics
J. Phys. Chem. B, Vol. 109, No. 41, 2005 19535 HA. These desirable properties make La3+-HA exhibit more potent photodamage ability on CT DNA than HA in both aerobic and anaerobic conditions, where the formation of 1O2 and other reactive radical species such as O2•-, OH•, and DNA•+ lead to the damage of DNA via both type I and type II mechanisms (see Scheme 1). In aerobic conditions, all four processes (processes 1-4 in Scheme 1) may take effect, while process 1 may predominate in anaerobic circumstances. Acknowledgment. This research was financially supported by the Natural Science Foundation of Jiangsu Education Department (Grant 2004191TSJB141), and Jiangsu Engineering Research Center for Bio-medical Function Material.
Figure 10. Chemiluminescent assay for photoinduced CT DNA damage sensitized by HA or La3+-HA. La3+-HA + CT DNA (a), HA + CT DNA (b), and CT DNA control (c).
SCHEME 1: Possible Pathways for the Photodamage of CT DNA Sensitized by La3+-HA
HA (9.03% binding sites destroyed) (Table 1). This kind of PDT effect initiated via the electron-transfer process from CT DNA to the triplet La3+-HA or HA, and the improved affinity between La3+-HA and CT DNA and the longer lifetime of triplet La3+-HA, might be responsible for its stronger PDT behavior in anaerobic condition. DNA has weak chemiluminescence; if it is damaged, the chemiluminescence will be intensified greatly.26 The chemiluminescence assay was also utilized to characterize the CT DNA damage photosensitized by HA and La3+-HA. Figure 10 illustrates the results of chemiluminescence assays on CT DNA damage. Comparing the chemiluminescence degrees of HA and La3+-HA systems, it is obvious that the photodamage of CT DNA induced by La3+-HA was about 2-fold stronger than that induced by HA under the same experimental conditions, consistent with the EB assay. 4. Conclusion In summary, La3+-HA exhibits an enhanced 1O2 generation quantum yield, a red-shifted absorption window, a greatly improved water solubility, a much lengthened triplet excited state lifetime, and an increased affinity to DNA with respect to
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