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Control of Photobleaching in Photodynamic Therapy Using the Photodecarbonylation Reaction of Ruthenium Phthalocyanine Complexes via Stepwise Two-Photon Excitation Kazuyuki Ishii,*,†,| Masahiko Shiine,‡ Yoshitaka Shimizu,‡ Shun-ichi Hoshino,‡ Hisaku Abe,‡ Kazuhiro Sogawa,§ and Nagao Kobayashi*,‡,⊥ Institute of Industrial Science, The UniVersity of Tokyo, 4-6-1 Komaba Meguro-ku, Tokyo 153-8505, Japan, Department of Chemistry, Graduate School of Science, Tohoku UniVersity, Sendai 980-8578, Japan, and Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku UniVersity, Sendai 980-8578, Japan ReceiVed: August 1, 2007; In Final Form: December 22, 2007
In this study, we have investigated the photochemical properties and photodynamic effects of ruthenium phthalocyanine (RuPc(CO)(Py)) and naphthalocyanine (RuNc(CO)(Py)) complexes. When a nanosecondpulsed laser is used, the photodecarbonylation of our Ru complexes efficiently proceeds via stepwise twophoton excitation, while the reaction yields are negligibly small when a continuous-wave (CW) laser is employed. The pulsed laser selective photodecarbonylation decreases the Q-band absorbance, which satisfies what the photodynamic therapy (PDT) requires of the photobleaching. For RuPc(CO)(Py), the photochemical reactions including both the photodecarbonylation and just photobleaching occur in HeLa cells in vitro. Toxicity and phototoxicity tests indicate that our RuPc(CO)(Py) and RuNc(CO)(Py) complexes in concentrations of 0.3-1 µM and 1-2 µM, respectively, are applicable as PDT agents. The phototoxicity is consistent with the photochemical properties of these complexes, namely, excited triplet lifetimes (10 and 4.8 µs for the Pc and Nc complexes, respectively) and singlet oxygen yields (0.48 and 0.35 for the Pc and Nc complexes, respectively). On the basis of these results, we propose a novel concept for achieving a greater depth of necrosis in PDT as follows: (1) PDT of upper cellular layers using CW-laser irradiation; (2) efficient photobleaching in upper cellular layers using pulsed dye-laser irradiation, which results in an increase in the therapeutic depth of red light; (3) PDT directed toward deeper tumor tissues using CW laser irradiation. In addition, these Ru complexes are promising as CO release agents for investigative biochemistry.
Introduction
CHART 1
Photodynamic therapy (PDT) is currently under intense study for the diagnosis, management, and treatment of various neoplasms, based on the combined use of the selective uptake of photosensitizers into malignant tissues and the local irradiation of these with visible or near-infrared (NIR) light.1-7 The first photosensitizer used was Photofrin, which is a complex mixture composed of a fraction of hematoporphyrin derivatives. Although Photofrin has been shown to be effective in the treatment of many cancers, it has disadvantages such as a small extinction coefficient at the radiation wavelength (630 nm) and cutaneous phototoxicity over a prolonged period of time. These disadvantages have prompted a search for the “second” and “third” generation photosensitizers in order to achieve high accumulation ratios between tumor and normal tissues and large extinction coefficients at longer wavelengths at which light can penetrate skin. In order to obtain intense absorption at longer wavelengths, various types of photosensitizers, including phthalocyanines * Authors to whom correspondence should be addressed. † The University of Tokyo. ‡ Department of Chemistry, Graduate School of Science, Tohoku University. § Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University. | Fax: +81-3-5452-6306. E-mail:
[email protected]. ⊥ Fax: +81-22-795-7719. E-mail:
[email protected].
(Pc’s), naphthalocyanines (Nc’s), benzoporphins, purpurins, chlorins, porphycenes, pheophorbides, bacteriochlorins, and bacteriopheophorbides, have been investigated.1-7 These compounds have intense absorption bands in the visible or NIR regions (the Q-bands), which increase the excitation probability. However, it has been suggested that a large extinction coefficient can be very detrimental to achieving greater depths of necrosis in PDT.6-9 That is, the photosensitizers that produce significant additional absorption in tumor tissues can induce considerable opacity in such tissues and, in fact, completely negate any advantage gained by the longer wavelength absorption, in comparison with the case of Photofrin. Therefore, the phenomenon of photobleaching is required for achieving PDT directed toward deeper tumor tissues since photodecomposition in upper cellular layers increases the therapeutic depth of the light. On the other hand, rapid photobleaching of the photosensitizers
10.1021/jp076118k CCC: $40.75 © 2008 American Chemical Society Published on Web 02/20/2008
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Figure 2. Time-courses of the photodecarbonylation reaction yields of RuPc(CO)(Py) (top) and RuNc(CO)(Py) (bottom) in pyridine upon 632.8 nm excitation (O: 1 mW CW He-Ne laser; b: 1 mW, 5 Hz pulsed dye-laser). Here, the absorbance at 632.8 nm was completed (∼0.1) in order to compare the Pc and Nc ligands. Figure 1. Electronic absorption and phosphorescence spectra of RuPc(CO)(Py) (top, solid line), RuPc(Py)2 (top, broken line), RuNc(CO)(Py) (bottom, solid line), and RuNc(Py)2 (bottom, broken line). Pyridine was used as the solvent for the electronic absorption measurements, while a 1:1 mixture of pyridine and toluene was employed as the glassy solvent for the phosphorescence measurements.
decreases the efficiency of PDT. Thus, a good balance between photobleaching and photostability under the PDT conditions is desirable; however, it is difficult to control photobleaching in biological environments. Recently, we have shown that the photodecarbonylation of a ruthenium(II) carbonyl octaethylporphyrin (RuOEP(CO)(Py)) complex occurs efficiently via stepwise two-photon excitation when using a nanosecond-pulsed laser of visible green light;10 however, ultraviolet light is required in the case of continuouswave (CW) of light. The photophysical properties are changeable before and after the photodecarbonylation. This has motivated us to prepare novel functional PDT photosensitizers based on pulsed laser selective photodecarbonylation. In this study, we investigate the photochemical properties and photodynamic effects of ruthenium phthalocyanine (RuPc(CO)(Py)) and naphthalocyanine (RuNc(CO)(Py)) complexes (Chart 1). By expanding the π-electron system, the excitation source of the photodecarbonylation has been converted to a nanosecondpulsed laser of red light that exhibits a high transparency through living tissue. It is noteworthy that the pulsed laser selective photodecarbonylation decreases the Q-band absorbance. Although the photochemical reaction is different from just photobleaching where all the absorption is lost, it satisfies what the PDT requires of the photobleaching, in terms of the decrease in absorbance at the Q-band peak () the excitation wavelength). That is, this Q-band absorbance is controllable by the pulsed laser irradiation. Since the photochemical reactions including both the photodecarbonylation and just photobleaching occur in HeLa cells in vitro and these Ru complexes show useful photochemical properties and phototoxicity toward them, we have proposed a novel concept for controlling photobleaching in PDT by the combined use of photobleaching with a nanosecond-pulsed laser and photodynamic treatment with a CW laser.
Experimental Section Instrumental Techniques. Electronic absorption spectra were measured with a Hitachi U-3410 spectrophotometer. NIR luminescence measurements were performed using a monochromator (JASCO CT-25CP) and a photomultiplier (Hamamatsu Photonics R5509-42), which was cooled at 193 K by a cold nitrogen gas flow system (Hamamatsu Photonics R6544-20).11 The photon signals amplified by a fast preamplifier (Stanford Research SR445) were measured by the single-photon counting method using a photon counter (Stanford Research SR400). A dye laser (Sirah CSTR-LG532-TRI-T) pumped with a Nd:YAG laser (Spectra Physics INDI 40; 532 nm, 7 ns fwhm), He-Ne red laser (NEC GLG5090; 632.8 nm), or diode laser (LDX Optronics LDX-2515-650; 650 nm) was employed as the excitation source. As a solvent, pyridine (Wako Pure Chemicals) was used for the electronic absorption measurements, while a 1:1 mixture of pyridine and spectral grade toluene (Nacalai Tesque Inc.) was employed for the phosphorescence measurements. Synthesis. Triruthenium dodecacarbonyl, H2Pc, and H2Nc were purchased from Aldrich Chemical Co. RuPc(Py)2 was prepared from H2Pc by the method described in the literature.12 RuPc(CO)(Py), RuNc(CO)(Py), and RuNc(Py)2 were synthesized with reference to the synthesis of Ru porphyrin complexes.10 The analytical data of these complexes are summarized in Supporting Information. Photodecarbonylation Reactions in Pyridine. For photochemical reactions, the concentrations of the samples were in the range of 2-5 µM, and the pyridine solutions were bubbled with nitrogen gas for 1 h beforehand. The photodecarbonylation reaction yields were determined by fitting the difference electronic absorption spectra before and after photoexcitation with the difference spectrum of the CO and bis(pyridine) complexes.10 In order to complete the cross section, a pin hole (diameter ∼ 2.5 mm) was inserted between the sample and the light source. Toxicity and Phototoxicity of Photosensitizers toward HeLa Cells. Our Ru complexes were encapsulated in liposomes for delivery to the HeLa cells. Small unilamellar liposomes of Ru complexes in L-R-dipalmitoylphosphatidylcholine (DPPC)
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TABLE 1: Photophysical Data of Ru Complexes Q-band/nm (10-4 )
λP/nma
τP/µsa
Ru complexes
in pyridine
in liposomes
293 K
77 K
293 K
77 K
Φ∆b,c
RuPc(CO)(Py) RuPc(Py)2 RuNc(CO)(Py) RuNc(Py)2
651 (21.5) 630 (8.2) 728.5 (34.2) 717 (21.9)
652
946 870 1094 971
939 860 1072 955
10 0.16 4.8 4.0
20 0.65 8.3 7.3
0.48 0.25 0.35 0.43
723
a In phosphorescence measurements, samples were deaerated by freeze-pump-thaw cycles. b Toluene solution containing 0.01 M pyridine was employed as a solvent; sample solutions were bubbled with oxygen gas. c Φ∆ values were determined by the use of (dihydroxy)tetra-tertbutylphthalocyaninatosilicon (Φ∆ ) 0.31).11b Since the decay profiles of singlet oxygen (1∆g) luminescence are independent of photosensitizers, the photon signals were counted at 4 µs (gate-width ) 5 µs) after the laser excitation in order to remove fluorescence influences.
CHART 2
were prepared using methods reported previously (Supporting Information).13,14 Monolayer cultures of HeLa were grown in Eagle’s minimal essential medium with 10% fetal bovine serum. The cultures were maintained at 37 °C in humidified 5% CO2 and 95% air.14,15 Toxicity and phototoxicity were investigated using methods reported previously.14 Suspensions of 2 × 103 HeLa cells in 100 µL of the medium were inoculated into each well of a 96well microplate. After culturing in a CO2 incubator for 24 h, the monolayer cultures of HeLa cells were treated with the photosensitizer at a concentration of 0.1-5 µM by adding 10 µL of PBS solutions containing 1.1-55 µM photosensitizers in liposomal dispersions. The photosensitizers were added to only 40 wells of the 96-well microplate in order to use the remaining wells as controls. After incubating for 20 h, the medium containing the photosensitizers was removed from each well, and 100 µL of fresh medium was added. Here, only in the case of the phototoxicity tests, the 40 wells treated with the photosensitizer were irradiated for 30 min using a dye-laser beam (10 mJ/pulse, 10 Hz), which was expanded by lenses (total fluence ∼ 5 J/cm2) in order to irradiate the 40 wells simultaneously from the bottom of the microplate. After incubation for 24 h, 10 µL of Cell Counting Kit-8 (CCK-8; Dojindo) solution was added into each well, and then they were cultured for ∼2 h.16 After the color reaction was completed, the absorbance at 450 nm for each well was measured with a microplate reader (BIO-RAD, MODEL 680). The toxicity and phototoxicity of the photosensitizers were evaluated by comparing the average absorbance data of the 40 treated wells with those of the untreated wells. Photochemical Reactions in HeLa Cells. Photochemical reactions of RuPc(CO)(Py) in HeLa cells were measured using the following procedure. Suspensions of 2.5 × 106 HeLa cells in 9 mL of the medium were inoculated into a 10 cm dish. After culturing in a CO2 incubator for 24 h, the monolayer cultures of HeLa cells were treated with Pc at a concentration of 5 µM by adding 1 mL of PBS solutions containing 50 µM Pc in liposomal dispersions. After incubating for 20 h, the medium
containing the photosensitizers was removed from the dish and repeatedly washed with PBS solutions in order to remove extracellular liposomes. Here, the decrease in the Q-band absorbance could not be confirmed when the pulsed dye-laser beam was expanded by lenses in order to irradiate the 10 cm dish simultaneously. This was due to the low photon flux density, which is inefficient for two-photon excitation. In order to maintain the high photon flux density of the pulsed dyelaser beam, ∼1 × 107 cells were harvested into a Pyrex tube (the diameter of the flat base is ∼3 mm) by the trypsinization of the monolayer. The Pyrex tube was subsequently centrifuged in a centrifuge tube to obtain a colorless supernatant fluid and blue-colored precipitate. Next, the HeLa cells treated with the photosensitizer were irradiated at 652 nm using a dye laser (20 mJ/pulse, 10 Hz) pumped with a Nd:YAG pulsed laser or a CW diode-laser (total fluence ∼ 1 W/cm2). After irradiation with red light, the Pc photosensitizer was extracted with excess dimethylformamide (DMF) from the blue-colored precipitate, and the absorbance at the Q-band was measured. Results and Discussion Electronic Absorption Spectra. The electronic absorption spectra in pyridine are shown in Figure 1, and the data are summarized in Table 1. In the case of the CO complexes, sharp and intense Q-absorption bands are observed at 651 and 728.5 nm for RuPc(CO)(Py) and RuNc(CO)(Py), respectively. On the other hand, the Q-absorption bands of the bis(pyridine) complexes are evidently different from those of their corresponding CO complexes. The Q-band peak (717 nm) of RuNc(Py)2 exhibits a blue-shift relative to that of RuNc(CO)(Py) (728.5 nm). In the case of the RuPc complexes, the Q-band broadens and blue-shifts (651 f 630 nm) by substituting the axial ligand CO with Py. To examine the electronic absorption spectra of the Ru complexes quantitatively, configuration interaction (CI) calculations were performed using the ZINDO/S Hamiltonian (Supporting Information).17-19 The spectral difference between the CO and bis(pyridine) complexes can be explained by the π-backdonation of the CO axial ligand. The π-back-donation of the
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Figure 3. Toxicity (O: broken line) and phototoxicity (b: solid line) response data of RuPc(CO)(Py) (left) and RuNc(CO)(Py) (right) toward HeLa cells in vitro. In the case of RuPc(CO)(Py), liposomes were prepared using a ratio of DPPC/RuPc(CO)(Py) ) 150. On the other hand, since RuNc(CO)(Py) tends to aggregate in liposomal dispersions, monomeric RuNc(CO)(Py) prepared by using DPPC/RuNc(CO)(Py) ) 1500 was employed for the toxicity and phototoxicity tests. The DPPC dependence on the electronic absorption spectra and phototoxicity are summarized in Supporting Information.
Figure 4. Electronic absorption spectra of RuPc(CO)(Py) extracted from HeLa cells with excess DMF.28 Solid and broken lines denote the absorption spectra before and after the pulsed dye-laser irradiation for 5 min, respectively. The inset shows the time-courses of decreases in the Q-band at 650 nm (O: CW diode-laser; b: pulsed dye-laser).
CO axial ligand stabilizes both the dπ (Ru) and π* (LUMOs of Pc or Nc) orbitals.20 However, the stabilization of the dπ orbitals is significantly larger than that of the π* orbitals. Therefore, the ππ* (Q-band origin) configurations exhibit a blue-shift; this is contrary to the red-shift expected in the metal-to-ligand charge transfer (MLCT; dπ (Ru) f π*(Pc)) configurations. In RuPc(CO)(Py), two intense Q-bands were calculated at 675 and 699 nm. Although these bands exhibit small splitting due to the axial Py ligand, this calculation result is consistent with the sharp, intense Q-band observed at 651 nm. These Q-bands mainly originate from the ππ* (a1ueg > 70%) configurations with a small contribution (∼5%) from the MLCT (dπ(Ru) f π*(Pc)). This is reasonably interpreted by the large energy difference between the MLCT and ππ* (a1ueg) configurations (>4000 cm-1). On the other hand, four intense transitions in the Q-band region (607, 623, 695, and 715 nm) were calculated for RuPc(Py)2, which resemble the broad Q-band observed at around 630 nm. This originates from strong admixtures between the red-shifted MLCT and blue-shifted ππ* configurations (the lowest energy difference is 1700 cm-1), the MLCT configurations admix to a small degree with the Q-band origin ππ*. Thus, the spectral changes in the Q-band can be reasonably interpreted by the π back-donation of the CO axial ligand. Luminescence Properties. Since PDT leading to biological damage usually involves the photochemical generation of singlet oxygen (1∆g) from the T1 photosensitizer and the subsequent oxidation of tissues by direct attack on biological substrates, luminescence measurements related to the photodynamic effects were performed at 293 and 77 K. The phosphorescence spectra at 293 K are shown in Figure 1, with data summarized in Table 1. In deaerated conditions, intense phosphorescence in the NIR region was observed at 946, 870, 1094, and 971 nm for RuPc(CO)(Py), RuPc(Py)2, RuNc(CO)(Py), and RuNc(Py)2, respectively. In the case of the CO complexes, the phosphorescent states are attributable to the 3(π,π) state in terms of the sharp spectra and relatively long excited-state lifetimes.21,22 The phosphorescence lifetime of RuPc(CO)(Py) (10 and 20 µs at 293 and 77 K, respectively) is longer than that of RuNc(CO)(Py) (4.8 and 8.3 µs at 293 and 77 K, respectively), as interpreted by the energy gap law.11c On the other hand, the phosphorescence properties are varied by the CO f Py substitution. RuPc(Py)2 exhibits a broad phosphorescence spectrum and a short phosphorescence lifetime (160 ns at 293 K), which evidently indicates the 3MLCT contribution toward the T1 RuPc(Py)2.12,21,22 This MLCT character in the T1 state resembles the Q-band properties. The phosphorescence lifetime of RuNc(Py)2 (4.0 and 7.3 µs at 293 and 77 K, respectively) is shorter than that of RuPc(CO)(Py); this is in contrast to the similarity in the T1 energies (10650 and 10470 cm-1 for RuPc(CO)(Py) and RuNc(Py)2, respectively). While the sharp phosphorescence spectrum of RuNc(Py)2 mainly originates from the 3(π,π) state, a small 3MLCT contribution shortens the T1 lifetime of RuNc(Py)2. To examine the phototoxic origin quantitatively, singlet oxygen yields (Φ∆) were investigated by measuring the singlet
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SCHEME 1 Concept for Achieving a Greater Depth of Necrosis in PDTa aInitially, tumor tissues treated with Ru complexes are irradiated by a CW diode-laser, which results in a photokill of the upper cellular layers (A f B). To eliminate the detrimental effect of the large extinction coefficient, the upper cellular layers are irradiated with a pulsed dye-laser (C). Since the efficient Q-band decrease in the upper cellular layers can increase the therapeutic depth of red light (D), PDT directed toward the deeper tumor tissues is achieved by irradiation with a CW diode-laser (E)
oxygen (1∆g) luminescence at around 1270 nm. From the phosphorescence spectra at 77 K, the T1 energies are evaluated as 10650, 11630, 9330, and 10470 cm-1 for RuPc(CO)(Py), RuPc(Py)2, RuNc(CO)(Py), and RuNc(Py)2, respectively. These energies are significantly higher than the singlet oxygen energies (7.84 × 103 cm-1), which indicates that the energy transfer process is exothermic. The Φ∆ values were evaluated as 0.48, 0.25, 0.35, and 0.43, for RuPc(CO)(Py), RuPc(Py)2, RuNc(CO)(Py), and RuNc(Py)2, respectively (Table 1). Although the Φ∆ value of RuPc(Py)2 is relatively small due to the short T1 lifetimes, the Φ∆ values of RuPc(CO)(Py), RuNc(CO)(Py), and RuNc(Py)2 are sufficiently high, and therefore, they are promising candidates for use as PDT photosensitizers. Photodecarbonylation Reactions. Figure 2 shows the time courses of the photodecarbonylation reaction yields in pyridine upon laser excitation (632.8 nm). The photodecarbonylation of RuPc(CO)(Py) and RuNc(CO)(Py) occurs efficiently upon pulsed dye-laser excitation (632.8 nm, 1 mW, fwhm ) 5-8 ns, 5 Hz). Thus, we have succeeded in converting the excitation source from green light to red light with high living body tissue transparency. The reaction yields with the CW He-Ne laser irradiation (632.8 nm, 1 mW) are considerably smaller (1028 photons/cm2 s.26,27 Since the T1 lifetimes (10 and 4.8 µs for RuPc(CO)(Py) and RuNc(CO)(Py), respectively) are significantly longer than the pulse width of our pulsed laser, the T1 state is considered to be an intermediate excited state, as shown in Chart 2. It is noteworthy that the pulsed laser-selective photodecarbonylation decreases the absorbance at the Q-band peak ( ) 2.15 × 105 f 5.20 × 104 at 651 nm for RuPc, and ) 3.42 × 105 f 1.06 × 105 at 728.5 nm for RuNc). This originates from the broadening or the shift of the Q-band due to the photodecarbonylation. Although these photochemical reactions are different from just photobleaching where all the absorption is lost, they satisfy what the PDT requires of the photobleaching, in terms of the decrease in absorbance at the Q-band peak () the excitation wavelength). In addition, the CO complexes exhibit useful photochemical properties such as long τT and large ΦD values. Thus, our CO complexes are promising as novel functional PDT photosensitizers capable of controlling the Q-band absorbance by the combined use of photodecarbonylation with the nanosecond-pulsed laser and photodynamic treatment with the CW laser. Toxicity and Phototoxicity toward HeLa Cells. In order to examine the photodynamic effects in the biological environ-
ment, the toxicity and phototoxicity toward HeLa cells in vitro were investigated for RuPc(CO)(Py) and RuNc(CO)(Py), as shown in Figure 3. The treatment of cultures with RuPc(CO)(Py) does not lead to a loss of cell validity in the absence of light at a Pc concentration of 0.3 µM, RuPc(CO)(Py) causes >85% destruction of dehydrogenase activity. These results indicate that RuPc(CO)(Py) has useful ability as a PDT photosensitizer at concentrations of 0.3-1 µM. Figure 3 (right) shows that the toxicity of RuNc(CO)(Py) is negligibly small at a concentration of 2 µM in the cultures. On the other hand, when RuNc(CO)(Py) is irradiated with a 724 nm dye-laser, high phototoxicity toward HeLa cells is observed at concentrations of >1 µM. Thus, RuNc(CO)(Py) exhibits the toxic and phototoxic characteristics applicable to PDT at concentrations of 1-2 µM. The photodynamic effect of RuPc(CO)(Py) is higher than that of RuNc(CO)(Py), while the number of moles of Ru complexes in a HeLa cell (1.8 × 10-16 and 1.6 × 10-16 mol/cell for RuPc(CO)(Py) and RuNc(CO)(Py), respectively) was independent of the Pc and Nc ligands; this was determined by extracting the Ru complexes with DMF from the blue-colored precipitate and measuring the absorbance at the Q-band. Since the Φ∆ and τp values of RuPc(CO)(Py) are larger than those of RuNc(CO)(Py), the high phototoxicity of RuPc(CO)(Py) can be interpreted on the basis of its higher ability to generate singlet oxygen. Photochemical Reactions in HeLa Cells. To confirm the photochemistry in the biological environment, the photochemical reactions of RuPc(CO)(Py) were typically examined in HeLa cells in vitro. Here, ∼1 × 107 HeLa cells were harvested into a Pyrex tube (the diameter of the flat base is ∼3 mm), and they were irradiated using a pulsed dye-laser or a CW diode-laser. Figure 4 shows the electronic absorption spectra of RuPc(CO)(Py) extracted from HeLa cells with excess DMF before and after the laser irradiation.28 The Q-band intensity dramatically decreases (
