ARTICLE pubs.acs.org/bc
Dual Signal-Responsive Liposomes for Temperature-Controlled Cytoplasmic Delivery Tomohiro Kaiden,† Eiji Yuba,† Atsushi Harada,† Yuichi Sakanishi,‡ and Kenji Kono*,† †
Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan ‡ Daicel Chemical Industries, Ltd., 2-1-4, Higashisakae, Ohtake, Hiroshima 739-0695, Japan
bS Supporting Information ABSTRACT: For production of a new type of functional liposome whose destabilization can be triggered by a combination of a temperature signal and acidic pH signal, we prepared liposomes modified with hyperbranched poly(glycidol) derivatives having N-isopropylamide and carboxyl groups. HeLa cells incubated with the dual signal-responsive liposomes encapsulating a water-soluble fluorescent dye pyranine at 28 °C displayed punctate fluorescence of pyranine, indicating that the liposomes were trapped in endosome. However, after heating at 45 °C for 15 min, the same cells exhibited diffuse fluorescence of pyranine, indicating that destabilization of the liposomes in endosome with an acidic environment in combination with the brief heating caused efficient transfer of the contents into cytosol. The dual signal-responsive liposomes might have usefulness for site-specific delivery of membrane-impermeable molecules, which exhibit bioactivities in the intracellular spaces, such as siRNA and proteins.
’ INTRODUCTION Recent advances in molecular biology and biotechnology have introduced highly efficient therapeutic approaches based on specific interaction to target molecules, as shown in typical examples of siRNA for specific gene silencing and protein inhibitors and antibodies of various kinds for targeted therapy. Many of their target molecules exist in intracellular spaces. Therefore, to maximize their therapeutic effects, accurate delivery of these bioactive molecules, not only to target diseased tissues, but also into their intracellular spaces where their target sites exist, are highly necessary. Various types of nanoparticles with stimuli-sensitive properties have been produced to increase local concentration of therapeutic molecules at the target tissues. Typical examples are temperature-sensitive liposomes, which release contents in response to elevated temperature.13 These temperature-sensitive liposomes encapsulating membrane-permeable anticancer drugs, such as doxorubicin, exhibited efficient tumor growth suppression upon mild heating of tumor sites.4,5 On the other hand, intracellular delivery of bioactive molecules, which cannot permeate cellular membranes, has been attempted using liposomes having pH-sensitive properties: so-called pH-sensitive liposomes.6,7 Liposomes are generally taken up by cells through endocytosis and are trapped in endosome, which contains a weakly acidic environment in its interior. Therefore, those with pH-sensitive properties generate abilities to release bioactive molecules and promote their transfer into cytosol.69 r 2011 American Chemical Society
On the basis of the functions of these temperature-sensitive and pH-sensitive liposomes, we attempted to develop a new type of liposome having a combination of these functions, which generate a membrane-destabilizing ability under conditions of mildly acidic pH with an elevated temperature. Their ability to respond to both pH and temperature signals might be useful for targeting tissue-specific intracellular delivery of membrane-impermeable molecules, because such dual-signal-responsive liposomes can destabilize and fuse with endosomes only when entrapped in endosomes of the target tissue, which has been mildly heated (Figure 1). Recently, we synthesized hyperbranched poly(glycidol)s (HPGs) having N-isopropylamide (NIPAM) and succinylate (Suc) groups, which are, respectively, temperature-sensitive and pH-sensitive groups.10 These polymers exhibited the dual-signalsensitive property; their characteristics changed from hydrophilic to hydrophobic in response to both pH decreasing and temperature increasing.10 For the present study, we incorporated these polymers onto stable egg yolk phosphatidylcholine (EYPC) liposomes to produce dual-signal-responsive liposomes. The pH-responsive and temperature-responsive properties of the HPGs-modified liposomes and their performance as intracellular delivery vehicles were investigated. Received: January 19, 2011 Revised: July 14, 2011 Published: August 23, 2011 1909
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Figure 1. Design of dual-signal-responsive liposomes whose destabilization can be controlled by temperature and acidic pH. Dual-signal-responsive liposomes are expected to deliver contents into the cytosol of cells of target tissues, which are locally heated.
’ EXPERIMENTAL PROCEDURES Materials. HPG with the polymerization degree of 40, which is designated as HPG40, was kindly donated by Daicel Chemical Industries, Ltd. (Osaka, Japan). Egg yolk phosphatidylcholine (EYPC) was kindly donated by NOF Co. (Tokyo, Japan). Pyranine, decylamine, and Triton X-100 were obtained from Tokyo Chemical Industries Ltd. (Tokyo, Japan). p-Xylene-bispyridinium bromide (DPX) and LysoTracker Red DND-99 were purchased from Molecular Probes (Oregon, USA). Lissamine rhodamine B-sulfonyl phosphatidylethanolamine (Rh-PE) was purchased from Avanti Polar Lipids (Birmingham, AL, USA). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride (DMT-MM), isopropylamine, and N-methylpyrrolidone were from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Succinic anhydride was obtained from Kishida Chemical Co., Ltd. (Osaka, Japan). Synthesis of NIPAM-Suc-HPG. NIPAM-Suc-HPGs were prepared as previously reported.10 A typical procedure was as follows. HPG40 (5.66 g) and an excessive amount of succinic anhydride (0.24 mol) were dissolved in pyridine (60 mL) and stirred at 65 °C for 7 h under an argon atmosphere. The polymer was washed with diethyl ether and was purified using a Sephadex LH-20 column with methanol to afford Suc-HPG40. The obtained Suc-HPG40 (0.82 g) was dissolved in N-methylpyrrolidone (8 mL), and DMT-MM (4.0 mmol) and isopropylamine (2.9 mmol) dissolved in N-methylpyrrolidone (1.2 mL) was added to the solution. The mixed solution was stirred at room temperature for 24 h under an argon atmosphere in the dark, and then, decylamine (0.48 mmol) dissolved in N-methylpyrrolidone
(1 mL) was added to the solution. The mixture was stirred at room temperature for 3 days under an argon atmosphere in the dark. To the reaction mixture, acetic acid (3 mL) was added and stirred for 1 h. The polymer was precipitated with water and washed with diethyl ether. Then, the polymer was purified using a Sephadex LH-20 column with methanol to afford NIPAM35Suc55-HPG. Compositions of polymers were estimated using 1H NMR. 1H NMR spectra for Suc-HPG40 and NIPAM35-Suc55HPG were shown in Supporting Information Figure S1 A and B, respectively. Integration of peaks for these spectra indicated that essentially no hydroxyl groups remained in Suc-HPG40 (Figure S1 A) and the molar ratio of NIPAM, Suc, and N-decylamide (DA) groups was 34.9/55.3/9.8 in NIPAM35-Suc55-HPG (Figure S1 B). Turbidity Measurements. Turbidity of NIPAM-Suc-HPGs dissolved in 10 mM phosphate and 150 mM NaCl solution (pH 5.07.4, 5 mg/mL) was measured at 700 nm using a Jasco model V-560 spectrophotometer equipped with a Peltier-type thermostatic cell holder coupled with an ETC-505T controller. The heating rate of the sample was 1.0 °C min1. The cloud points were taken as the initial break points in the resulting transmittance versus temperature curves. Preparation of Liposomes. To a dry, thin membrane of EYPC (7 mg) and polymer (7 mg) was added 500 μL of aqueous 35 mM pyranine, 50 mM DPX, and 50 mM phosphate solution (pH 7.4), and the mixture was sonicated for 10 min using a bathtype sonicator. The liposome suspension was further hydrated by freezing and thawing repeatedly, and was extruded through a polycarbonate membrane with a pore size of 100 nm. The liposome suspension was dialyzed against 10 mM phosphate and 140 mM NaCl buffer (pH 7.4) at 4 °C for 2 days. Then, 1910
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Figure 2. Preparation of NIPAM-Suc-HPG. DMT-MM and NMP represent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride and N-methylpyrrolidone, respectively.
liposome was ultracentrifuged twice at 55 000 rpm for 2 h to remove free pyranine and free polymer from the pyranine-loaded liposomes. Pyranine Release from Liposomes. Release of pyranine from liposomes was measured as previously reported.8,11 Pyranine fluorescence was quenched by DPX inside of the liposomes, but it exhibits intense fluorescence when released from the liposome. Polymer-modified liposomes encapsulating pyranine and DPX were suspended in 10 mM phosphate and 140 mM NaCl buffer of varying pHs (lipid concentration: 2.0 105 M) at varying temperatures, and fluorescence intensity at 512 nm of the suspension was followed with excitation at 416 nm using a spectrofluorometer (Jasco FP-6200). The percent release of pyranine from liposomes was defined as releaseð%Þ ¼ ðFt Fi Þ=ðFf Fi Þ 100 where Fi and Ft mean the initial and intermediary fluorescence intensities of the liposome suspension, respectively. Ff is the fluorescence intensity of the liposome suspension after addition of Triton X-100 (final concentration: 0.1%). Intracellular Distribution of Liposomes. The pyranineloaded liposomes containing Rh-PE were prepared as described above except that DPX was not included in the pyranine solution and Rh-PE (0.1 mol %) was included as a liposome component. HeLa cells (2 105 cells) cultured in DMEM supplemented with 10% FBS and antibiotics for 2 days were washed with phosphate-buffered saline (PBS), and then incubated in DMEM without serum (500 μL). The pyranine-loaded liposomes (0.6 mM, 500 μL) were added gently to the cells and incubated for 4 h at 28 °C. After the incubation, the cells were washed with PBS three times and heated at 45 °C for 15 min. Confocal laser scanning microscopic (CLSM) analysis of these cells was performed using LSM 5 EXCITER (Carl Zeiss Co. Ltd.). The cells without the 15 min heating at 45 °C were also observed using CLSM (λex = 485 nm). For inhibition of endosomal acidification, cells were preincubated with 100 μM chloroquine for 30 min before the incubation with liposomes.
’ RESULTS AND DISCUSSION Preparation of Polymers for Liposome Modification. Hyperbranched poly(glycidol)s having NIPAM and Suc groups and
Table 1. Characterization of NIPAM-Suc-HPGsa molar ratiob polymer
NIPAM
Sue
DA
NIPAM0-Suc89-HPG
0
88.9
11.1
NIPAM35-Suc55-HPG NIPAM56-Suc33-HPG
34.9 56.4
55.3 32.9
9.8 10.7
NIPAM64-Suc27-HPG
63.5
26.6
9.9
NIPAM72-Suc17-HPG
71.9
17.3
10.8
NIPAM64-Suc36-HPGcont
63.8
36.2
0
a
Suc-HPG40 was obtained by reacting HPG40 completely with excess succinic anhydride. And then NIPAM and DA groups were incorporated to Suc-HPG40 by condensation of a part of Suc groups with isopropylamine and decylamine. b Molar ratios of NIPAM, Suc, and DA groups in the polymers were evaluated based on 1H NMR (see Supporting Information).
decyl chains, which are anchors for their fixation onto liposomes, were prepared as presented in Figure 2 using HPG with the number average polymerization degree of 40 as previously reported.10 The pH-sensitive and temperature-sensitive properties of the HPGs having NIPAM and Suc groups (NIPAM-SucHPGs) are dependent on their compositions.10 Therefore, HPGbased polymers of five kinds having varying ratios of NIPAM and Suc groups were prepared for modification of liposomes (Table 1). We introduced decyl groups to about 10 unit % of the chain terminals of the polymers as anchors for their incorporation to liposomes. In addition, NIPAM-Suc-HPG having the NIPAM/Suc ratio of 64/36 without decyl groups, which is designated as NIPAM64-Suc36-HPGcont, was synthesized as a control without anchors. First, we examined pH-sensitive and temperature-sensitive properties of the NIPAM-Suc-HPGs by the detection of phase separation of their aqueous solutions (Figure 3). Figure 3A,B shows the optical transmittance of the NIPAM65-Suc35-HPGcont and NIPAM56-Suc33-HPG solutions, respectively, at various pH values as a function of temperature. The solutions of these polymers were transparent at neutral pH in the experimental temperature region, indicating that their chains were highly hydrated irrespective of temperature at neutral pH. However, 1911
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Figure 3. Temperature and pH-sensitive phase transition of NIPAM-Suc-HPGs. Temperature dependence of transmittance for solutions of NIPAM64Suc36-HPGcont (A) and NIPAM56-Suc33-HPG (B) at various pHs. (C) Cloud points of NIPAM64-Suc36-HPGcont (open circles), NIPAM35-Suc55-HPG (squares), NIPAM56-Suc33-HPG (closed circles), NIPAM64-Suc27-HPG (triangles), and NIPAM72-Suc17-HPG (diamonds) as a function of pH.
these polymer solutions exhibited a temperature-dependent decrease in transmittance at pH 56, indicating that these polymers changed their characteristic from hydrophilic to hydrophobic depending on temperature in the weakly acidic pH region. These polymers showed the water-solubility change under similar temperature and pH regions, probably because these polymers have similar ratios of NIPAM units to succinylate units, which contribute, respectively, to temperature and pH sensitivities. Additionally, it is readily apparent that NIPAM56Suc33-HPG changed its water-solubility with temperature gradually in broad temperature regions at weakly acidic pH compared to NIPAM65-Suc35-HPGcont. For NIPAM56-Suc33-HPG, hydrophobic decyl chains connected to the polymer backbone might make its conformation more compact. Then, the polymer chains taking on a compact conformation would be unable to interact mutually as efficiently as NIPAM65-Suc35-HPGcont taking on an expanded conformation, resulting in the transition in the broad temperature region for the anchor-carrying polymer. We defined the cloud point of the polymer solution as the temperature at which the transmittance became 50%. Figure 3C shows cloud points of NIPAM-Suc-HPGs with various NIPAM/ Suc ratios as a function of pH. Generally, the polymers exhibited cloud points between 20 and 60 °C. However, the pH region in which these polymers showed the cloud point tends to increase concomitantly with increase of the NIPAM/Suc ratio, indicating that their pH-sensitive and temperature-sensitive properties are controllable by adjusting their NIPAM/Suc ratio. Dual Signal-Responsive Behaviors of Polymer-Modified Liposomes. To investigate production of dual signal-responsive liposomes, we examined the effect of modification of stable EYPC liposomes with the NIPAM-Suc-HPGs. On the basis of the result of Figure 3, we chose three kinds of HPG-based polymers with different NIPAM/Suc unit ratios, NIPAM56Suc33-HPG, and NIPAM72-Suc17-HPG, as well as NIPAM-free NIPAM0-Suc90-HPG, to elucidate how the NIPAM/Suc unit ratio of the polymer affects pH- and temperature-sensitive properties of their modified liposomes. The liposomes were prepared by dispersing a mixture of egg yolk phosphatidylcholine (EYPC) and polymer in a phosphate solution containing watersoluble fluorescent dye pyranine and its quencher DPX and subsequent extrusion through a polycarbonate membrane with a pore size of 100 nm. The liposomes were purified by dialysis and subsequent ultracentrifugation. Their content release behaviors were investigated by following the fluorescence of pyranine, which become highly fluorescent when released from the liposome.11,12 We confirmed that pyranine release from the
unmodified EYPC was very low and that it was affected only slightly by pH and temperature under experimental conditions (see Figure S2). However, the liposomes modified with these polymers exhibited pyranine release depending on the ambient pH and temperature (Figure 4). The liposomes modified with NIPAM0-Suc90-HPG showed significant pH-responsive content release, which was significantly promoted below pH 5.5 and which achieved complete release at pH 4.5 (Figure 4A,D). However, these liposomes exhibited fundamentally equivalent pH-dependent release behavior irrespective of ambient temperature, indicating that they are responsive only to ambient pH. On the other hand, the liposomes modified with NIPAM56Suc33-HPG exhibited significant pH-responsive contents release at 37 and 45 °C, but they only slightly release the contents at 10 °C (Figure 4B,E). This fact indicates that these liposomes have sensitivity to both pH and temperature. Consequently, their function can be dually controlled by ambient pH and temperature. The NIPAM72-Suc17-HPG-modified liposomes showed the temperature-dependent enhancement of content release even at neutral pH, although they exhibited pH-dependent enhancement of content release below pH 6.0 to some degree (Figure 4C,F). Apparently, modification with the polymer having a high content of NIPAM moieties abolished pH-responsive properties of the liposomes and provided rather temperature-responsive properties to the liposomes. Because the NIPAM56-Suc33-HPG-modified liposomes exhibited excellent response to both temperature and pH, we further examined the temperature-dependent enhancement of contents release at neutral pH and at pH 5.5, which respectively corresponds to the extracellular pH and the endosome pH.13 As Figure 5 depicts, contents released from the liposomes were suppressed effectively, even at 50 °C under neutral pH condition, indicating that the liposomes retained pyranine at that pH. Similarly, under a weakly acidic condition, the liposomes retained contents less than 25 °C, but at greater than 30 °C, the liposomes enhanced the content release. On the basis of the result of Figure 2C, the NIPAM56-Suc33-HPG might change its character from hydrophilic to hydrophobic around 30 °C at pH 5.5. Therefore, the dehydrated polymer chains attached to the liposome surface might interact strongly with the liposome membrane and induce perturbation of the liposome membrane. Control of Cytoplasmic Delivery by Dual Signal-Responsive Liposomes. We examined intracellular delivery mediated by the dual signal-responsive NIPAM56-Suc33-HPG-modified liposomes. The liposome membrane was labeled with Rh-PE to detect the location of liposomes in the cell. Plain EYPC 1912
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Figure 4. Typical profiles of pyranine release from liposomes modified with NIPAM0-Suc90-HPG (A), NIPAM56-Suc33-HPG (B), and NIPAM72Suc17-HPG (C) at pH 7.4 (diamonds), pH 5.0 (circles), and pH 4.0 (triangles) at 10 °C (open symbols) or 45 °C (closed symbols). pH-Dependence of pyranine release from liposomes modified with NIPAM0-Suc90-HPG (D), NIPAM56-Suc33-HPG (E), and NIPAM72-Suc17-HPG (F). Percent release after 15 min incubation at 10 °C (diamonds), 37 °C (squares), or 45 °C (triangles) was shown.
Figure 5. (A) Typical profiles of pyranine release from NIPAM56-Suc33-HPG-modified liposomes at pH 7.4 (diamonds) and pH 5.5 (squares) at 10 °C (open symbols) or 45 °C (closed symbols). (B) Temperature dependence of pyranine release at pH 7.4 (diamonds) and pH 5.5 (squares). Percent release after 10 min incubation was shown.
liposomes labeled with Rh-PE and loaded with pyranine were also examined as a control. These liposomes were incubated with HeLa cells for 4 h at 28 °C, where the NIPAM56-Suc33-HPGmodified liposomes were stable, even at weakly acidic pH (Figure 5B), then washed with PBS three times to remove free liposomes. The cells were additionally incubated in the culture medium at 28 or 45 °C for 15 min and observed using CLSM (Figure 6). As presented in Figure 6A, cells treated with the plain
liposome at 28 °C displayed punctate fluorescence of Rh-PE and pyranine in the peripheral region of the cells at the same locations, suggesting that the liposomes retaining pyranine were trapped in early endosomes, which exist near the cellular surface. Because the cells were treated with the liposomes at 28 °C, the cellular activity might have been reduced. Consequently, internalization of the liposomes into the cells was limited largely to that taking place within the cellular membrane periphery. When 1913
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Rh-PE fluorescence remained located around the cellular membrane. This fact indicates that pyranine molecules were released from liposomes and were introduced into cytosol upon heat application, although the liposomes were still in endosomes near the cellular membrane (Figure 6D). On the basis of Figure 5B, NIPAM56-Suc33-HPG chains attached onto the liposome surface strongly destabilize lipid membranes under a weakly acidic environment at temperatures higher than 35 °C. Therefore, it is likely that the polymer chains can cause significant destabilization of both the liposome and endosome membranes, enabling pyranine molecules to enter into the cytosol. Compared to the plain liposomes, the NIPAM56-Suc33-HPG-modified liposomes seemed to exhibit a tendency to remain in the peripheral region of the cellular membrane. The highly hydrated polymer chains covering the liposome surface may affect cellular processes, such as endocytosis, although the reason is unclear. We also examined intracellular delivery mediated by the dual signal-responsive liposomes in the presence of chloroquine, which inhibits acidification of endosome.14 As Figure 6F shows, cellular diffuse pyranine fluorescence was suppressed in the presence of chloroquine, indicating that a weakly acidic environment is required for temperature-induced triggering of liposome destabilization. These results demonstrate that destabilization of the dual signal-responsive liposomes can be triggered by mild heat application only for those trapped in weakly acidic compartments. To confirm superiority of the dual signal-responsive liposomes, we further examined intracellular delivery using pH-sensitive but temperature-insensitive NIPAM0-Suc90-HPG-modified liposomes (Figure 4D) via the same incubation procedures. HeLa cells treated with the NIPAM0-Suc90-HPG-modified liposomes encapsulating pyranine exhibited mainly punctate fluorescence of pyranine around the periphery of cellular membrane irrespective of the temperature of the 15 min incubation (Supporting Information Figure S3). Because the cells were incubated with the liposomes for 4 h at 28 °C, which is much lower than the physiological temperature, reduced cellular activity might suppress the efficient lowering of pH in endosome. Considering that destabilization of NIPAM0-Suc90-HPG-modified liposomes was induced below pH 5.0 (Figure 4D), the liposomes trapped in endosome might not be efficiently destabilized, resulting in the limited extent of pyranine transfer into cytosol. This result suggests another merit of the dual signal-responsive liposomes, whose destabilization is efficiently induced through the synergy of protonated carboxyl groups and dehydrated NIPAM groups on the polymer chains. Figure 6. CLSM images of HeLa cells treated with pyranine-loaded plain (A,B) and NIPAM56-Suc33-HPG-modified (CF) liposomes labeled with Rh-PE in the absence (AD) or presence (E,F) of 100 μM chloroquine. Cells (2 105 cells) were incubated in the serum-free medium containing liposomes (0.3 mM) for 4 h at 28 °C and washed with phosphate-buffered saline three times. Then, the cells were additionally incubated at 28 °C (A,C,E) or 45 °C (B,D,F) for 15 min and were observed with CLSM. Bars represent 20 μm.
the cells were heated to 45 °C, the cells displayed the same fluorescence, indicating that the heat application did not affect the location of pyranine molecules in the cells (Figure 6B). Similarly, cells treated with the dual signal-responsive NIPAM56-Suc33-HPG-modified liposomes at 28 °C mainly showed fluorescence around the cell surface (Figure 6C). However, when heated at 45 °C, the cells displayed the entirely different fluorescence of pyranine, which diffused in the cytosol, although
’ CONCLUSION We developed a new type of functional liposome whose destabilization can be dually controlled by pH and temperature using pH-sensitive and temperature-sensitive NIPAM-SucHPGs. The dual signal-responsive liposomes destabilized only when mildly heated under mildly acidic conditions. Therefore, only the liposomes taken up by cells and trapped in acidic compartments can be destabilized by mild heating. These liposomes destabilized in acidic endosome caused the transfer of contents into cytosol. To date, temperature-sensitive liposomes of various types have been developed. Their functions are focused only on temperature-induced control of drug release. Therefore, these liposomes may not be useful for delivery of membrane-impermeable molecules, such as siRNA and proteins, whose active sites exist in the intracellular space, because a large 1914
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fraction of the contents may be released from the liposomes in the extracellular space of the heated area. In contrast, the dual signal-responsive liposomes could release the contents only when trapped in the endosome of the cells in the heated area. Therefore, the dual signal-responsive liposomes might solve such problems of conventional temperature-sensitive liposomes and increase the efficacy and reliability of therapies based on these bioactive molecules. Attempts to adjust the temperature region in which the liposomes show response and to achieve sharp and drastic response upon signal applications are currently underway.
(13) Mellman, I. (1996) Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575–625. (14) Misinzo, G., Delputte, P. L., and Nauwynck, H. J. (2008) Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells. J. Virol. 82, 1128–1135.
’ ASSOCIATED CONTENT
bS
1
H NMR spectra of polymers (Figure S1), pyranine release from plain liposomes (Figure S2), and CLSM images of HeLa cells treated with NIPAM0-Suc90HPG-modified liposomes (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information.
’ AUTHOR INFORMATION Corresponding Author
*Tel: +81-722-54-9330; Fax: +81-722-54-9330; E-mail: kono@ chem.osakafu-u.ac.jp.
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