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Hypoxic condition-selective upconversion via triplet–triplet annihilation based on POSS-core dendrimer complexes
Bioorganic & Medicinal Chemistry 21 (2013) 2678–2681
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Hypoxic condition-selective upconversion via triplet–triplet annihilation based on POSS-core dendrimer complexes Kazuo Tanaka, Hiroshi Okada, Wataru Ohashi, Jong-Hwan Jeon, Kenichi Inafuku, Yoshiki Chujo ⇑ Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
a r t i c l e
i n f o
Article history: Received 5 February 2013 Revised 19 March 2013 Accepted 21 March 2013 Available online 29 March 2013 Keywords: Upconversion Triplet–triplet annihilation POSS-core dendrimer Dissolved oxygen
a b s t r a c t The influence on the efficiencies of the triplet–triplet annihilation (TTA)-supported upconversion by oxygen under biomimetic conditions was investigated. From the solution containing the dendrimer complexes based on polyhedral oligomeric silsesquioxane (POSS)-core dendrimer with the Pt complex of octaethylporphyrin (PtOEP) and anthracene in PBS, the fluorescence emission of anthracene depending on the dissolved oxygen (DO) concentrations via the TTA-supported upconversion was obtained with the excitation light at 540 nm. In particular, we observed strong emission only under hypoxic conditions. In addition, it was found that the emission intensity via TTA-supported upconversion can be reversibly regulated by the DO concentrations in the solution. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Photon upconversion, in which the shorter-wavelength light is generated than that of the incident light, under biological conditions has recently gathered much attention as a key phenomenon for improving the signal to noise ratios in the imaging with the optical probes.1 By using lanthanoid nanocrystals with the excitation light in the red or near infrared light, it was demonstrated that autofluorescence from the samples can be suppressed.2 Furthermore, upconversion is attempted to apply for the low-invasive photo-triggered drug release.3 Longer-wavelength lights are favorable due to higher permeability to active the light-responsive materials at the deep spot inside bodies. Indeed, the nanoparticle-based host materials have been developed for photodynamic therapy3a or molecular release3b via upconversion. Upconversion can occur via TTA, in which one singlet-excited molecule can be generated from two triplet-excited molecules.4 Particularly, there is the significant advantage for applying TTA to biological conditions that TTA-supported upconversion can proceed under continuous wave-irradiation even such as sunlight.5 Furthermore, the excitation and emission wavelength can be tuned by modulating the sensitizer and the emitter pairs.6 Although the great advances on TTA-supported upconversion have been received in material applications, there are only a few examples to show the feasibility of the TTA-supported upconversion in biotechnology.1 We have recently reported the environmentalresponsive photon upconversion under biomimetic conditions.7 ⇑ Corresponding author. Tel.: +81 75 383 2604; fax: +81 75 383 2605. E-mail address: [email protected] (Y. Chujo). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.03.029
Visible light (540 nm) was converted to the light from near UV light (>390 nm) in the buffer. Moreover, by modulating the encapsulation ability of the dendrimers with the sensitizer and the emitter, the changes of upconversion efficiencies were observed. To extend the applicability of TTA-supported upconversion in biological and biomedical fields, the establishment of the stimuli-responsiveness of emission efficiency should be required. It was reported that the critical reduction of the DO level was detected in the active solid tumors.8 Thus, the hypoxia-specific upconversion is promised to be a fundamental technique to develop the tumor-selective photo-triggered drug release and drug activation.9 Herein, we report the hypoxia-selective upconversion via TTA under biomimetic conditions. We prepared the dendrimer complex based on the second generation (G2) of POSS-core dendrimer with PtOEP and anthracene according to our previous report.7 By employing PtOEP as an oxygen-sensitive unit,10 the DO level-dependent emission via the TTA-supported upconversion was observed in PBS. We received the strong emission only under the hypoxic conditions. Furthermore, the upconversion emission can be modulated reversibly by changing the DO level in the solutions. This is the first example, to the best of our knowledge, to offer the regulation method of upconversion emission by biologically-significant factor under biomimetic conditions. 2. Experimental section 2.1. General UV–vis absorption spectra were obtained at 25 °C using 1 cm path length cell with a SHIMADZU UV-3600UV–vis-NIR
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K. Tanaka et al. / Bioorg. Med. Chem. 21 (2013) 2678–2681
spectrophotometer. The fluorescence emission under excitation at 540 nm was monitored using a Perkin Elmer LS50B at 25 °C using 1 cm path length cell with a 480-nm cut-off filter. DO levels were determined with a HORIBA OM-51 DO meter. The excitation bandwidth was 15 nm. The emission bandwidth was 5 nm. Synthesis of the dendrimer was executed according to the previous reports.11 2.2. Sample preparations The aqueous solution of 1 mM G2 POSS-core dendrimer (50 lL) and the THF solution of 100 lM PtOEP (5 lL) were added to the DMSO solution of 1 mM anthracene (50 lL). Subsequently, water (345 lL) and 10-times condensed PBS stocked solution (50 lL) were added. The DO level in the solution was modulated by changing the mixing ratios with N2- and O2-bubbled water (DO concentrations: 2.2 and 24.1 mg/mL, respectively). The optical properties of the resulting solutions were measured within 10 min. 2.3. Switching experiment To the bulk solution containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS, O2 and N2 gasses were alternatively passed for 3 min at room temperature. Then, the plots represent the emission intensity at 420 nm obtained from the solution with the excitation light at 540 nm.
Uup ¼ UðAn;em;DMSO;376exÞ Iup =IðAn;em;DMSO;376exÞ eðAn;376Þ =eðcomplex;540Þ ccomplex =cAn ðnðH2 OÞ =nðDMSOÞ Þ2 S1nm =S15nm E376 =E540
Here, U(An,em,DMSO,376ex) is the quantum yield of the fluorescence emission from anthracene with the excitation at 376 nm in DMSO determined to be 0.18 as an absolute value with an integrating sphere. I is the emission area calculated from the spectrum, e is the molar extinct coefficient, c is the concentration, n is the refractive index of each solvent, S is the light amplitude in each slit width, and E is the light amplitude in each wavelength. To determine the quantum yields for each step, initially, we defined the quantum yield of upconversion (Uup) as Eq. 2:
Uup ¼ UISC Usens UTTA UðAn;em;DMSO;376exÞ
UISC ¼ Uphos Iðphos;waterÞ =Iðphos;DMSOÞ eð540;DMSOÞ =eð540;waterÞ S5nm =S15nm
Usens ¼ 1 ðI=I0 Þ
S1
ΦTTA
Φem T1
Φsens
S0
ð4Þ
Here, the integration of the phosphorescence from PtOEP was represented as I0 in the absence and I in the presence of anthracene.
T1 λex (540 nm)
ð3Þ
Here, Uphos is the quantum yield of phosphorescence of PtOEP. I is the phosphorescence intensity from PtOEP in each solvent, e is the molar extinct coefficient of the solution at 540 nm in each solvent.
ΦISC
S1
λ em (390-450 nm)
Sensitizer = PtOEP
Fluorophore = Anthracene
S0
R
R R Si O Si O Si O Si O O O O O O Si OO Si Si O Si R R R R O NH2 N N 2 H
R
R=
ð2Þ
Here, the efficiency of the generation of the triplet-excited PtOEP (UISC) was evaluated as a relative value from the phosphorescence intensity according to Eq. 3. From the decrease of the emission band from the triplet-excited state of PtOEP, the efficiency for sensitizing (Usens) was calculated according to Eq. 4. The quantum yield of upconversion (Uup) was calculated as a relative value compared to the fluorescence emission of anthracene with the excitation at 376 nm. Thus, the efficiency of TTA (UTTA) was obtained.
2.4. Fluorescence measurements with the dendrimer complexes Fluorescence measurements were executed with the aqueous solutions containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS at 25 °C with the excitation at 540 nm passing through a 480 nm cut-off filter. Samples were bubbled with nitrogen for 1 h before the measurements. The quantum yields were determined by comparing to the fluorescence intensities in the spectra of anthracene with the excitation at 376 nm in DMSO or water according to Eq. 1. 7
ð1Þ
N
N Pt
N
N
Anthracene PtOEP
G2 POSS-core dendrimer Scheme 1. The energy diagram of the upconversion via TTA and chemical structures of the compounds used in this study.
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Figure 1. DO-dependent fluorescence changes of the solutions containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS at 25 °C with the excitation light at 540 nm passing through a 480 nm cut-off filter. The DO levels in the samples were 2.2 mg/mL (magenta) and 24.1 mg/mL (blue), respectively.
3. Results and discussion The chemical structures of the molecules used in this study are shown in Scheme 1. POSS has a strong hydrophobicity, and a wide variety of aromatic and dye molecules can be retained into the POSS-core dendrimers by adsorbing onto the core.11d Consequently, the encapsulation by POSS-core dendrimers enhances the compatibility of the encapsulated molecules and maintains the good dispersibility under biological conditions. PtOEP as a triplet sensitizer for visible-light absorber and anthracene as an emitter of UV light were simultaneously adsorbed onto the POSS core in the water-soluble G2 POSS-core dendrimer.7 PtOEP works as a photosensitizer for exciting anthracene to the triplet-excited state by the light irradiation at longer-wavelength light. Subsequently, singlet-excited anthracene can be generated from two triplet-excited anthracene molecules via TTA.5b As a result, fluorescence emission from anthracene can be obtained (390 nm). Moreover, the triplet-excited state of PTOEP would be decayed by oxygene.10 Thus, we expected to observe the oxygen-sensitive upconversion from the dendrimer complexes involving PtOEP and anthracene. The dendrimer complexes were prepared as follows: The aqueous solution of 1 mM G2 POSS-core dendrimer (50 lL) and the THF solution of 100 lM PtOEP (5 lL) were mixed, and then the DMSO solution of 1 mM anthracene (50 lL) was added. Water (345 lL) and subsequently 10 PBS stocked solution (50 lL) were added. The DO level in the solution was modulated by changing the mixing ratios with N2- and O2-bubbled water. The optical properties of the resulting solutions were measured within 10 min. The excitation light was at 540 nm passed through the cutoff filter below 480 nm to eliminate the direct excitation of anthracene by a half wavelength light generated inevitably from xenon lamps. The quantum yields in each step were determined as a relative value described in the Section 2. The fluorescence emission from anthracene was observed in the range of 400–450 nm. Initially, we measured upconversion emission from the sample containing the dendrimer complexes in PBS (Fig. 1). The lowest DO sample (2.2 mg/mL) showed the largest emission with the peaks at 400, 420, and 450 nm corresponded to the fluorescent emission from anthracene. By increasing the DO level, the intensity decreased. The relationship between the DO level and the emission intensity is illustrated in Figure 2. Below 13.2 mg/mL of DO level, the quenching efficiency slowly increased by increasing the DO level in the solution. In contrast, over 18.6 mg/mL of DO level, the quenching of the upconversion emission was greatly enhanced.
Figure 2. Stern–Volmer plots of the upconversion emission by increasing the DO concentration. The emission intensity of the sample with 2.4 mg/mL DO concentration was used as an initial emission intensity (I0) to calculate the quenching ratio.
In the previous report, it was revealed that the encapsulation by the POSS-core dendrimer can inhibit photo-degradation of the encapsulated dyes.11d These results suggest that the accessibility of oxygen could be decreased by the POSS core. In this study, it is likely that the POSS-core dendrimer could disturb the deactivation of the triplet excited states of the chromophores by oxygen molecules. These data clearly indicate that the emission efficiency of the TTA-supported upconversion using the dendrimer complexes can be controlled by the DO dependency. In particular, it has been reported that the DO level in the hypoxia in the solid tumour is approximately 2 mg/mL.8 On the other hand, the DO level is maintained around 20 mg/mL in the normal cells.8 Therefore, the threshold is located at the suitable region for discriminating the normal and tumour cells with the upconversion emission. To confirm that the quenching of the upconversion emission was caused by oxygen, we calculated the efficiencies and quantum yields of each step to the emission as a relative value (Table 1). Here, we defined the quantum yield of upconversion (Uup) as the Eq. 1:
Uup ¼ UISC Usens UTTA UAn;em
ð1Þ
The quantum yield of fluorescence emission from anthracene (UAn,em) was determined to be 0.18 as an absolute value with an integrating sphere. The efficiency of the generation of the tripletexcited PtOEP was approximately 0.9.7 From the decrease of the emission band of the triplet-excited state of PtOEP observed between 600 and 750 nm by the addition of anthracene, the efficiency for sensitizing (Usens) was determined as 0.21. Quantum yield of upconversion (Uup) was calculated to be 0.051 104 as a relative value compared to the fluorescence emission of anthracene with the excitation at 376 nm because of extremely-low efficiencies of the generation of the triplet excited state of PtOEP. From these values, the efficiency of TTA (UTTA) with PtOEP was obtained as 0.027. As same procedure, the quantum yields were evaluated
Table 1 Optical properties of DO-dependent upconversion emissionsa
a
DO concentration (mg/mL)
Usens
UTTA
UISC (103)
Uup (104)
2.2 7.8 13.2 18.6 24.1
0.21 0.23 0.18 0.19 0.10
0.027 0.022 0.027 0.012 0.019
0.90 0.84 0.82 0.85 0.80
0.051 0.043 0.039 0.021 0.017
Procedures and conditions are described in the Section 2.
K. Tanaka et al. / Bioorg. Med. Chem. 21 (2013) 2678–2681
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applicable for developing fundamental and essential technologies to establish the tumor-specific drug activation via the photon upconversion. Acknowledgement This research was partly supported by the Japan Securities Scholarship Foundation (for K.T.). References and notes
Figure 3. Switching of the upconversion emission by changing the DO concentration. To the bulk solution containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS, O2 (odd steps) and N2 (even steps) gasses were alternatively passed for 3 min. The plots represent the emission intensity at 420 nm obtained from the solution with the excitation light at 540 nm.
from the solutions with various DO levels. By increasing the DO level of the solutions, the UISC, Usens, and UTTA significantly decreased. These facts indicate that oxygen should quench the triplet-excited states of PtOEP and anthracene, resulting in the decreases of upconversion emissions. Finally, to evaluate the reversibility of the modulation of the upconversion efficiency, the emission intensity was monitored by changing the DO level of the solutions (Fig. 3). By alternatively bubbling O2 and N2 gases to the sample, the emission intensities at 420 nm were monitored. Correspondingly, the fluorescence emission decreased and increased by the O2 and N2 bubbling, respectively. These results suggest that this system is applicable for realizing the site-specific upconversion depending on the DO level by automatically switching the upconversion efficiency. In particular, hypoxia-selective upconversion can be expected. 4. Conclusion The dendrimer complexes with metalloporphyrin and anthracene were prepared, and the influence of the TTA-supported upconversion from visible to UV light on the DO level was evaluated. It was found that the upconversion efficiency was regulated by the DO level. In particular, the significant emission was observed only from the hypoxic samples. Furthermore, the upconversion emission can be dynamically modulated by changing the DO level. Our dendrimer complexes might be promised to be the light source for activating the photo-triggered drugs only in hypoxia under the longer-wavelength light irradiation. These results could be
1. (a) Wohnhaas, C.; Turshatov, A.; Mailänder, V.; Lorenz, S.; Baluschev, S.; Miteva, T.; Landfester, K. Macromol. Biosci. 2011, 11, 772; (b) Liu, Q.; Yang, T.; Feng, W.; Li, F. J. Am. Chem. Soc. 2012, 134, 5390. 2. (a) Sivakumar, S.; Diamente, P. R.; van Veggel, F. C. J. M. Chem. Eur. J. 2006, 12, 5878; (b) Kamimura, M.; Miyamoto, D.; Saito, Y.; Soga, K.; Nagasaki, Y. Langmuir 2008, 24, 8864; (c) Soukka, T.; Rantanen, T.; Kuningas, K. Ann. N.Y. Acad. Sci. 2008, 1130, 188; (d) Zako, T.; Nagata, H.; Terada, N.; Sakono, M.; Soga, K.; Maeda, M. J. Mater. Sci. 2008, 43, 5325; (e) Wang, L.; Li, Y. Chem. Commun. 2006, 2557; (f) Wan, C.; Xia, T.; Becker, H. C.; Zewail, A. H. Chem. Phys. Lett. 2005, 412, 158; (g) Lu, H.; Yi, G.; Zhao, S.; Guo, L. H.; Cheng, J. J. Mater. Chem. 2004, 14, 1336. 3. Carling, C.-J.; Nourmohammadian, F.; Boyer, J.-C.; Branda, N. R. Angew. Chem., Int. Ed. 2010, 49, 3782. 4. (a) Singh-Rachford, T. N.; Castellano, F. N. Coord. Chem. Rev. 2010, 254, 2560; (b) Simon, Y.; Weder, C. J. Mater. Chem. 2012, 22, 20817; (c) Cheng, Y. Y.; Fückel, B.; MacQueen, R. W.; Khoury, T.; Clady, R. G. C. R.; Schulze, T. F.; Ekins-Daukes, N. J.; Crossley, M. J.; Stannowski, B.; Lips, K.; Schmidt, T. W. Energy Environ. Sci. 2012, 5, 6953; (d) Yi, X.; Zhao, J.; Sun, J.; Guo, S.; Zhang, H. Dalton Trans. 2013, 42, 2062. 5. (a) Islangulov, R. R.; Kozlov, D. V.; Castellano, F. N. Chem. Commun. 2005, 3776; (b) Islangulov, R. R.; Lott, J.; Weder, C.; Castellano, F. N. J. Am. Chem. Soc. 2007, 129, 12652; (c) Baluschev, S.; Miteva, T.; Yakutkin, V.; Nelles, G.; Yasuda, A.; Wegner, G. Phys. Rev. Lett. 2006, 97, 143903; (d) Merkel, P. B.; Dinnocenzo, J. P. J. Lumin. 2009, 129, 303; (e) Ynag, P.; Wu, W.; Zhao, J.; Huang, D.; Yi, X. J. Mater. Chem. 2012, 22, 20273. 6. (a) Miteva, T.; Yakutkin, V.; Nelles, G.; Baluschev, J. New J. Phys. 2008, 10, 103002; (b) Singh-Rachford, T. N.; Haefele, A.; Ziessel, R.; Castellano, F. N. J. Am. Chem. Soc. 2008, 130, 16164; (c) Chen, H. C.; Hung, C. Y.; Wang, K. H.; Chen, H. L.; Fann, W. S.; Chien, F. C.; Chen, P.; Chow, T. J.; Hsu, C. P. Chem. Commun. 2009, 4064. 7. Tanaka, K.; Inafuku, K.; Chujo, Y. Chem. Commun. 2010, 46, 4378. 8. (a) Höckel, M.; Schlenger, K.; Hockel, S.; Aral, B.; Schaffer, U.; Vaupel, P. Int. J. Cancer 1998, 79, 365; (b) Carmeliet, P.; Ng, Y.-S.; Nuyens, D.; Theilmeier, G.; Brusselmans, K.; Cornelissen, I.; Ehler, E.; Kakkar, V. V.; Stalmans, I.; Mattot, V.; Perriard, J.-C.; Dewerchin, M.; Flameng, W.; Nagy, A.; Lupu, F.; Moons, L.; Collen, D.; D’Amore, P. A.; Shima, D. T. Nature 1998, 394, 485. 9. Tanabe, K.; Harada, H.; Narazaki, M.; Tanaka, K.; Inafuku, K.; Komatsu, H.; Ito, T.; Yamada, H.; Chujo, Y.; Matsuda, T.; Hiraoka, M.; Nishimoto, S. J. Am. Chem. Soc. 2009, 131, 15982. 10. (a) Wu, W.; Wu, W.; Ji, S.; Guo, H.; Zhao, J. Dalton Trans. 2011, 40, 5953; (b) Amao, Y.; Tabuchi, Y.; Yamashita, Y.; Kimura, K. Eur. Polym. J. 2002, 38, 675; (c) Amao, Y.; Miyashita, T.; Okura, I. React. Funct. Polym. 2001, 47, 49; (d) Gillanders, R. N.; Tedford, M. C.; Crilly, P. J.; Bailey, R. T. Anal. Chim. Acta 2004, 502, 1. 11. (a) Tanaka, K.; Jeon, J.-H.; Inafuku, K.; Chujo, Y. Bioorg. Med. Chem. 2012, 20, 915; (b) Tanaka, K.; Murakami, M.; Jeon, J.-H.; Chujo, Y. Org. Biomol. Chem. 2012, 10, 90; (c) Tanaka, K.; Kitamura, N.; Naka, K.; Morita, M.; Inubushi, T.; Chujo, M.; Nagao, M.; Chujo, Y. Polym. J. 2009, 41, 287; (d) Tanaka, K.; Inafuku, K.; Naka, K.; Chujo, Y. Org. Biomol. Chem. 2008, 6, 3899; (e) Jeon, J.-H.; Tanaka, K.; Chujo, Y. RSC Adv. 2013, 3; (f) Kitamura, N.; Hiraoka, T.; Tanaka, K.; Chujo, Y. Bioorg. Med. Chem. 2012, 20, 4668; (g) Tanaka, K.; Kitamura, N.; Chujo, Y. Bioorg. Med. Chem. 2012, 20, 96.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Hypoxic condition-selective upconversion via triplet–triplet annihilation based on POSS-core dendrimer complexes Kazuo Tanaka, Hiroshi Okada, Wataru Ohashi, Jong-Hwan Jeon, Kenichi Inafuku, Yoshiki Chujo ⇑ Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
a r t i c l e
i n f o
Article history: Received 5 February 2013 Revised 19 March 2013 Accepted 21 March 2013 Available online 29 March 2013 Keywords: Upconversion Triplet–triplet annihilation POSS-core dendrimer Dissolved oxygen
a b s t r a c t The influence on the efficiencies of the triplet–triplet annihilation (TTA)-supported upconversion by oxygen under biomimetic conditions was investigated. From the solution containing the dendrimer complexes based on polyhedral oligomeric silsesquioxane (POSS)-core dendrimer with the Pt complex of octaethylporphyrin (PtOEP) and anthracene in PBS, the fluorescence emission of anthracene depending on the dissolved oxygen (DO) concentrations via the TTA-supported upconversion was obtained with the excitation light at 540 nm. In particular, we observed strong emission only under hypoxic conditions. In addition, it was found that the emission intensity via TTA-supported upconversion can be reversibly regulated by the DO concentrations in the solution. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Photon upconversion, in which the shorter-wavelength light is generated than that of the incident light, under biological conditions has recently gathered much attention as a key phenomenon for improving the signal to noise ratios in the imaging with the optical probes.1 By using lanthanoid nanocrystals with the excitation light in the red or near infrared light, it was demonstrated that autofluorescence from the samples can be suppressed.2 Furthermore, upconversion is attempted to apply for the low-invasive photo-triggered drug release.3 Longer-wavelength lights are favorable due to higher permeability to active the light-responsive materials at the deep spot inside bodies. Indeed, the nanoparticle-based host materials have been developed for photodynamic therapy3a or molecular release3b via upconversion. Upconversion can occur via TTA, in which one singlet-excited molecule can be generated from two triplet-excited molecules.4 Particularly, there is the significant advantage for applying TTA to biological conditions that TTA-supported upconversion can proceed under continuous wave-irradiation even such as sunlight.5 Furthermore, the excitation and emission wavelength can be tuned by modulating the sensitizer and the emitter pairs.6 Although the great advances on TTA-supported upconversion have been received in material applications, there are only a few examples to show the feasibility of the TTA-supported upconversion in biotechnology.1 We have recently reported the environmentalresponsive photon upconversion under biomimetic conditions.7 ⇑ Corresponding author. Tel.: +81 75 383 2604; fax: +81 75 383 2605. E-mail address: [email protected] (Y. Chujo). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.03.029
Visible light (540 nm) was converted to the light from near UV light (>390 nm) in the buffer. Moreover, by modulating the encapsulation ability of the dendrimers with the sensitizer and the emitter, the changes of upconversion efficiencies were observed. To extend the applicability of TTA-supported upconversion in biological and biomedical fields, the establishment of the stimuli-responsiveness of emission efficiency should be required. It was reported that the critical reduction of the DO level was detected in the active solid tumors.8 Thus, the hypoxia-specific upconversion is promised to be a fundamental technique to develop the tumor-selective photo-triggered drug release and drug activation.9 Herein, we report the hypoxia-selective upconversion via TTA under biomimetic conditions. We prepared the dendrimer complex based on the second generation (G2) of POSS-core dendrimer with PtOEP and anthracene according to our previous report.7 By employing PtOEP as an oxygen-sensitive unit,10 the DO level-dependent emission via the TTA-supported upconversion was observed in PBS. We received the strong emission only under the hypoxic conditions. Furthermore, the upconversion emission can be modulated reversibly by changing the DO level in the solutions. This is the first example, to the best of our knowledge, to offer the regulation method of upconversion emission by biologically-significant factor under biomimetic conditions. 2. Experimental section 2.1. General UV–vis absorption spectra were obtained at 25 °C using 1 cm path length cell with a SHIMADZU UV-3600UV–vis-NIR
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spectrophotometer. The fluorescence emission under excitation at 540 nm was monitored using a Perkin Elmer LS50B at 25 °C using 1 cm path length cell with a 480-nm cut-off filter. DO levels were determined with a HORIBA OM-51 DO meter. The excitation bandwidth was 15 nm. The emission bandwidth was 5 nm. Synthesis of the dendrimer was executed according to the previous reports.11 2.2. Sample preparations The aqueous solution of 1 mM G2 POSS-core dendrimer (50 lL) and the THF solution of 100 lM PtOEP (5 lL) were added to the DMSO solution of 1 mM anthracene (50 lL). Subsequently, water (345 lL) and 10-times condensed PBS stocked solution (50 lL) were added. The DO level in the solution was modulated by changing the mixing ratios with N2- and O2-bubbled water (DO concentrations: 2.2 and 24.1 mg/mL, respectively). The optical properties of the resulting solutions were measured within 10 min. 2.3. Switching experiment To the bulk solution containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS, O2 and N2 gasses were alternatively passed for 3 min at room temperature. Then, the plots represent the emission intensity at 420 nm obtained from the solution with the excitation light at 540 nm.
Uup ¼ UðAn;em;DMSO;376exÞ Iup =IðAn;em;DMSO;376exÞ eðAn;376Þ =eðcomplex;540Þ ccomplex =cAn ðnðH2 OÞ =nðDMSOÞ Þ2 S1nm =S15nm E376 =E540
Here, U(An,em,DMSO,376ex) is the quantum yield of the fluorescence emission from anthracene with the excitation at 376 nm in DMSO determined to be 0.18 as an absolute value with an integrating sphere. I is the emission area calculated from the spectrum, e is the molar extinct coefficient, c is the concentration, n is the refractive index of each solvent, S is the light amplitude in each slit width, and E is the light amplitude in each wavelength. To determine the quantum yields for each step, initially, we defined the quantum yield of upconversion (Uup) as Eq. 2:
Uup ¼ UISC Usens UTTA UðAn;em;DMSO;376exÞ
UISC ¼ Uphos Iðphos;waterÞ =Iðphos;DMSOÞ eð540;DMSOÞ =eð540;waterÞ S5nm =S15nm
Usens ¼ 1 ðI=I0 Þ
S1
ΦTTA
Φem T1
Φsens
S0
ð4Þ
Here, the integration of the phosphorescence from PtOEP was represented as I0 in the absence and I in the presence of anthracene.
T1 λex (540 nm)
ð3Þ
Here, Uphos is the quantum yield of phosphorescence of PtOEP. I is the phosphorescence intensity from PtOEP in each solvent, e is the molar extinct coefficient of the solution at 540 nm in each solvent.
ΦISC
S1
λ em (390-450 nm)
Sensitizer = PtOEP
Fluorophore = Anthracene
S0
R
R R Si O Si O Si O Si O O O O O O Si OO Si Si O Si R R R R O NH2 N N 2 H
R
R=
ð2Þ
Here, the efficiency of the generation of the triplet-excited PtOEP (UISC) was evaluated as a relative value from the phosphorescence intensity according to Eq. 3. From the decrease of the emission band from the triplet-excited state of PtOEP, the efficiency for sensitizing (Usens) was calculated according to Eq. 4. The quantum yield of upconversion (Uup) was calculated as a relative value compared to the fluorescence emission of anthracene with the excitation at 376 nm. Thus, the efficiency of TTA (UTTA) was obtained.
2.4. Fluorescence measurements with the dendrimer complexes Fluorescence measurements were executed with the aqueous solutions containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS at 25 °C with the excitation at 540 nm passing through a 480 nm cut-off filter. Samples were bubbled with nitrogen for 1 h before the measurements. The quantum yields were determined by comparing to the fluorescence intensities in the spectra of anthracene with the excitation at 376 nm in DMSO or water according to Eq. 1. 7
ð1Þ
N
N Pt
N
N
Anthracene PtOEP
G2 POSS-core dendrimer Scheme 1. The energy diagram of the upconversion via TTA and chemical structures of the compounds used in this study.
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Figure 1. DO-dependent fluorescence changes of the solutions containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS at 25 °C with the excitation light at 540 nm passing through a 480 nm cut-off filter. The DO levels in the samples were 2.2 mg/mL (magenta) and 24.1 mg/mL (blue), respectively.
3. Results and discussion The chemical structures of the molecules used in this study are shown in Scheme 1. POSS has a strong hydrophobicity, and a wide variety of aromatic and dye molecules can be retained into the POSS-core dendrimers by adsorbing onto the core.11d Consequently, the encapsulation by POSS-core dendrimers enhances the compatibility of the encapsulated molecules and maintains the good dispersibility under biological conditions. PtOEP as a triplet sensitizer for visible-light absorber and anthracene as an emitter of UV light were simultaneously adsorbed onto the POSS core in the water-soluble G2 POSS-core dendrimer.7 PtOEP works as a photosensitizer for exciting anthracene to the triplet-excited state by the light irradiation at longer-wavelength light. Subsequently, singlet-excited anthracene can be generated from two triplet-excited anthracene molecules via TTA.5b As a result, fluorescence emission from anthracene can be obtained (390 nm). Moreover, the triplet-excited state of PTOEP would be decayed by oxygene.10 Thus, we expected to observe the oxygen-sensitive upconversion from the dendrimer complexes involving PtOEP and anthracene. The dendrimer complexes were prepared as follows: The aqueous solution of 1 mM G2 POSS-core dendrimer (50 lL) and the THF solution of 100 lM PtOEP (5 lL) were mixed, and then the DMSO solution of 1 mM anthracene (50 lL) was added. Water (345 lL) and subsequently 10 PBS stocked solution (50 lL) were added. The DO level in the solution was modulated by changing the mixing ratios with N2- and O2-bubbled water. The optical properties of the resulting solutions were measured within 10 min. The excitation light was at 540 nm passed through the cutoff filter below 480 nm to eliminate the direct excitation of anthracene by a half wavelength light generated inevitably from xenon lamps. The quantum yields in each step were determined as a relative value described in the Section 2. The fluorescence emission from anthracene was observed in the range of 400–450 nm. Initially, we measured upconversion emission from the sample containing the dendrimer complexes in PBS (Fig. 1). The lowest DO sample (2.2 mg/mL) showed the largest emission with the peaks at 400, 420, and 450 nm corresponded to the fluorescent emission from anthracene. By increasing the DO level, the intensity decreased. The relationship between the DO level and the emission intensity is illustrated in Figure 2. Below 13.2 mg/mL of DO level, the quenching efficiency slowly increased by increasing the DO level in the solution. In contrast, over 18.6 mg/mL of DO level, the quenching of the upconversion emission was greatly enhanced.
Figure 2. Stern–Volmer plots of the upconversion emission by increasing the DO concentration. The emission intensity of the sample with 2.4 mg/mL DO concentration was used as an initial emission intensity (I0) to calculate the quenching ratio.
In the previous report, it was revealed that the encapsulation by the POSS-core dendrimer can inhibit photo-degradation of the encapsulated dyes.11d These results suggest that the accessibility of oxygen could be decreased by the POSS core. In this study, it is likely that the POSS-core dendrimer could disturb the deactivation of the triplet excited states of the chromophores by oxygen molecules. These data clearly indicate that the emission efficiency of the TTA-supported upconversion using the dendrimer complexes can be controlled by the DO dependency. In particular, it has been reported that the DO level in the hypoxia in the solid tumour is approximately 2 mg/mL.8 On the other hand, the DO level is maintained around 20 mg/mL in the normal cells.8 Therefore, the threshold is located at the suitable region for discriminating the normal and tumour cells with the upconversion emission. To confirm that the quenching of the upconversion emission was caused by oxygen, we calculated the efficiencies and quantum yields of each step to the emission as a relative value (Table 1). Here, we defined the quantum yield of upconversion (Uup) as the Eq. 1:
Uup ¼ UISC Usens UTTA UAn;em
ð1Þ
The quantum yield of fluorescence emission from anthracene (UAn,em) was determined to be 0.18 as an absolute value with an integrating sphere. The efficiency of the generation of the tripletexcited PtOEP was approximately 0.9.7 From the decrease of the emission band of the triplet-excited state of PtOEP observed between 600 and 750 nm by the addition of anthracene, the efficiency for sensitizing (Usens) was determined as 0.21. Quantum yield of upconversion (Uup) was calculated to be 0.051 104 as a relative value compared to the fluorescence emission of anthracene with the excitation at 376 nm because of extremely-low efficiencies of the generation of the triplet excited state of PtOEP. From these values, the efficiency of TTA (UTTA) with PtOEP was obtained as 0.027. As same procedure, the quantum yields were evaluated
Table 1 Optical properties of DO-dependent upconversion emissionsa
a
DO concentration (mg/mL)
Usens
UTTA
UISC (103)
Uup (104)
2.2 7.8 13.2 18.6 24.1
0.21 0.23 0.18 0.19 0.10
0.027 0.022 0.027 0.012 0.019
0.90 0.84 0.82 0.85 0.80
0.051 0.043 0.039 0.021 0.017
Procedures and conditions are described in the Section 2.
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applicable for developing fundamental and essential technologies to establish the tumor-specific drug activation via the photon upconversion. Acknowledgement This research was partly supported by the Japan Securities Scholarship Foundation (for K.T.). References and notes
Figure 3. Switching of the upconversion emission by changing the DO concentration. To the bulk solution containing 100 lM G2 POSS-core dendrimer, 100 lM anthracene, 10 lM PtOEP, 10% DMSO, and 1% THF in PBS, O2 (odd steps) and N2 (even steps) gasses were alternatively passed for 3 min. The plots represent the emission intensity at 420 nm obtained from the solution with the excitation light at 540 nm.
from the solutions with various DO levels. By increasing the DO level of the solutions, the UISC, Usens, and UTTA significantly decreased. These facts indicate that oxygen should quench the triplet-excited states of PtOEP and anthracene, resulting in the decreases of upconversion emissions. Finally, to evaluate the reversibility of the modulation of the upconversion efficiency, the emission intensity was monitored by changing the DO level of the solutions (Fig. 3). By alternatively bubbling O2 and N2 gases to the sample, the emission intensities at 420 nm were monitored. Correspondingly, the fluorescence emission decreased and increased by the O2 and N2 bubbling, respectively. These results suggest that this system is applicable for realizing the site-specific upconversion depending on the DO level by automatically switching the upconversion efficiency. In particular, hypoxia-selective upconversion can be expected. 4. Conclusion The dendrimer complexes with metalloporphyrin and anthracene were prepared, and the influence of the TTA-supported upconversion from visible to UV light on the DO level was evaluated. It was found that the upconversion efficiency was regulated by the DO level. In particular, the significant emission was observed only from the hypoxic samples. Furthermore, the upconversion emission can be dynamically modulated by changing the DO level. Our dendrimer complexes might be promised to be the light source for activating the photo-triggered drugs only in hypoxia under the longer-wavelength light irradiation. These results could be
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