Remote-Controlled Release of Singlet Oxygen by the Plasmonic Heating of Endoperoxide-Modified Gold Nanorods: Towards a Paradigm Change in Photodynamic Therapy

Article abstract of DOI:10.1002/anie.201510064

The photodynamic therapy of cancer is contingent upon the sustained generation of singlet oxygen in the tumor region. However, tumors of the most metastatic cancer types develop a region of severe hypoxia, which puts them beyond the reach of most therapeutic protocols. More troublesome, photodynamic action generates acute hypoxia as the process itself diminishes cellular oxygen reserves, which makes it a self-limiting method. Herein, we describe a new concept that could eventually lead to a change in the 100 year old paradigm of photodynamic therapy and potentially offer solutions to some of the lingering problems. When gold nanorods with tethered endoperoxides are irradiated at 808 nm, the endoperoxides undergo thermal cycloreversion, resulting in the generation of singlet oxygen. We demonstrate that the amount of singlet oxygen produced in this way is sufficient for triggering apoptosis in cell cultures. EPT sees the light: When gold nanorods with tethered endoperoxides are irradiated with near-infrared light, the endoperoxides undergo thermal cycloreversion, resulting in the generation of singlet oxygen. The amount of singlet oxygen generated by these nanocomposites is sufficient for triggering apoptosis in cell cultures.

Full text of DOI:10.1002/anie.201510064

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201510064
German Edition: DOI: 10.1002/ange.201510064
Photodynamic Therapy
Remote-Controlled Release of Singlet Oxygen by the Plasmonic
Heating of Endoperoxide-Modified Gold Nanorods: Towards
a Paradigm Change in Photodynamic Therapy
Safacan Kolemen, Tugba Ozdemir, Dayoung Lee, Gyoung Mi Kim, Tugce Karatas,
Juyoung Yoon,* and Engin U. Akkaya*
Abstract: The photodynamic therapy of cancer is contingent
upon the sustained generation of singlet oxygen in the tumor
region. However, tumors of the most metastatic cancer types
develop a region of severe hypoxia, which puts them beyond
the reach of most therapeutic protocols. More troublesome,
photodynamic action generates acute hypoxia as the process
itself diminishes cellular oxygen reserves, which makes it a self-
limiting method. Herein, we describe a new concept that could
eventually lead to a change in the 100 year old paradigm of
photodynamic therapy and potentially offer solutions to some
of the lingering problems. When gold nanorods with tethered
endoperoxides are irradiated at 808 nm, the endoperoxides
undergo thermal cycloreversion, resulting in the generation of
singlet oxygen. We demonstrate that the amount of singlet
oxygen produced in this way is sufficient for triggering
apoptosis in cell cultures.
accumulation in tumors, this non-invasive, or minimally
invasive, treatment protocol, with tolerable side effects and
[6]
a bonus of enhanced immune response, has tremendous
therapeutic potential. However, the full promise of PDT has
not been realized except perhaps for some niche applications
[
7]
such as superficial lesions. The limited applicability is not
necessarily due to the lack of optimal sensitizers or smart
[8–15]
delivery/activation processes;
the problem unfortunately
lies at the core of the PDT paradigm. First, even at the
optimum wavelengths, the tissue penetration of light is very
[16]
ineffective beyond the first few millimeters. The second
issue is oxygen concentration. Most tumors develop a hypoxic
region, and this is more common in aggressive metastatic
[17]
tumors. Such hypoxic tissues are highly resistant to chemo-
[18]
therapy and radiotherapy.
PDT, on the other hand, is
inhibited even more severely, because normoxic oxygen
concentrations are essential for effective singlet-oxygen
generation. Furthermore, even in normal tissues, the PDT
process itself decreases the cellular oxygen concentration,
T
he photodynamic therapy (PDT) of cancer has been
considered a promising therapeutic approach for decades.
[1]
Principal requirements for photodynamic action were estab-
lished by the experiments of Raab and von Tappeiner at the
[
2]
turn of the 20th century. Based on their work, which was
[
3,4]
followed up by many others,
a photosensitizer that can be
excited by light in the visible, but more preferably in the red
or near-IR region of the spectrum could sensitize ground-
state molecular oxygen and generate a short-lived, cytotoxic
1
reactive oxygen species, that is, singlet oxygen (O : D ). This
2
g
process is inherently regioselective, as the singlet-oxygen
generation will take place only in the region at which the light
beam is directed. In combination with the enhanced perme-
[
5]
ation and retention (EPR) effect, which leads to sensitizer
[
*] Dr. S. Kolemen, Dr. T. Ozdemir, Prof. E. U. Akkaya
UNAM-Institute of Material Science and Nanotechnology
Bilkent University
Ankara 06800 (Turkey)
E-mail: eua@fen.bilkent.edu.tr
D. Lee, G. M. Kim, Prof. J. Yoon
Department of Chemistry and Nano Science
Ewha Womans University
Seoul 120-750 (Korea)
E-mail: jyoon@ewha.ac.kr
T. Karatas, Prof. E. U. Akkaya
Department of Chemistry, Bilkent University
Ankara 06800 (Turkey)
Figure 1. New photodynamics concept. Top: Synthesis of the targeted
anthracene endoperoxide derivative (EPT1) for gold nanorod function-
alization. The PEG linker enhances the water solubility of both the
nanorods and the anthracene derivative. Bottom: Plasmonic heating of
the gold nanorods at near-IR wavelengths leads to thermal cyclo-
reversion of the tethered endoperoxides yielding singlet oxygen.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1
002/anie.201510064.
3
606
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 3606 –3610

Angewandte
Communications
Chemie
[
19]
thus inhibiting the therapeutic process.
Clearly, in most
should yield nanorods that can release singlet oxygen upon
irradiation with near-IR light as a result of plasmonic heating.
applications, PDT becomes a self-limiting methodology.
Herein, we propose that if singlet oxygen could be
generated directly in the desired region by remotely con-
trolled mechanisms, all of these issues would be successfully
circumvented.
Endoperoxides are the most reliable chemical source of
singlet oxygen. Endoperoxides of naphthalene, anthracene,
and a few other arenes were shown to generate singlet oxygen
Singlet-oxygen generation in organic or aqueous solvents
through the decomposition of endoperoxides within a biolog-
[28–30]
ical context has been reported previously.
However, our
work is the first ever to demonstrate a viable process that
could replace PDT in cell cultures and uses tissue-penetrating
near-IR irradiation.
Kinetic stability at ambient temperature (or 378C) is
important. The half-lives of anthracene 9,10-endoperoxides
[20]
upon warming, with good chemical yields.
parameters for endoperoxide cycloreversion versus oxidative
The kinetic
[31]
are typically years at room temperature, but when heated,
they decompose rapidly, mostly by cycloreversion. With these
considerations, we targeted the synthesis of a thiol-terminated
and water-soluble 9,10-disubstituted anthracene, EPT1
(Figure 1; for details of its synthesis, see the Supporting
Information). Endoperoxide 2 can be prepared by the
reaction of compound 1 with oxygen gas bubbled through
the DCM/THF solution in the presence of methylene blue as
a photosensitizer under irradiation with a broadband white
lamp (200 W). Then, compound 2 was subjected to a DCC
coupling with amine-PEG-thiol to yield EPT1.
[21]
decomposition have been studied, and there are reported
examples of relatively stable endoperoxides that cleanly
undergo cycloreversion upon heating, generating singlet
oxygen.
Whereas there are other methods for remote-controlled
for our proof of principle, we chose to make use
of the plasmonic heating of metallic nanoparticles, specifically
that of gold nanorods. Based on literature precedence, and
targeting an aspect ratio of four, it is possible to prepare gold
nanorods with strong near-IR absorption (longitudinal peak).
Then, appropriate functionalization with endoperoxides
[
22–24]
[25,26]
heating,
[27]
Figure 2. Thermal cycloreversion of EPT1. a) Absorbance at one of the anthracene peaks (404 nm) after heating EPT1 for 30 min at the indicated
temperatures. b) Thermolysis of EPT1 in DMSO at 958C for 6 h. Inset: Absorbance of EPT1 in DMSO at 378C. Samples were heated for 6 h.
c) When EPT1 is heated in HEPES buffer (pH 7.2, 20 mm), the typical anthracene spectral signature is observed. d) Decrease in absorbance of
DPBF in DMSO in the presence of EPT1 upon heating at 708C and reappearance of the parent anthracene absorption bands.
Angew. Chem. Int. Ed. 2016, 55, 3606 –3610
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 3607

Angewandte
Communications
Chemie
Our first investigation was a chemical assessment of the
decomposition–cycloreversion process. We demonstrated that
upon heating in DMSO or HEPES buffer, the endoperoxide
cleanly regenerates the parent anthracene, as confirmed by
the typical electronic absorption spectrum of anthracene
incubated with EPT1–GNR or the control compound GNR–
PEG (GNRs with a PEG-SH ligand) for 24 h. Cryo-TEM
imaging shows that the EPT1–GNRs are located inside the
cells within vacuoles (see the Supporting Information, Fig-
ure S5). As an additional control, a singlet-oxygen-quencher
azide (10 mm) was added to the cell media in one series. To
detect singlet-oxygen generation, cells were stained with
(
Figure 2). Next, it was equally important to demonstrate that
singlet oxygen is among the products of the cycloreversion.
By using the selective singlet-oxygen trap 1,3-diphenyliso-
benzofuran (DPBF), we were able to detect singlet-oxygen
formation (Figure 2d) during the thermal decomposition of
the endoperoxide in DMSO, as the trap absorbance decreased
with a concomitant increase in the intensities of the character-
istic anthracene peaks.
ꢀ
Cyto-ID oxidative-stress detection reagents for 30 min after
À2
irradiation with an 808 nm NIR laser (2.0 Wcm ) for 10 min,
and then the cell images were acquired.
For the other component of the therapeutic agent, gold
nanorods with the desired characteristics (40 nm, aspect ratio:
[27]
4
) were prepared following literature precedence.
TEM
images of the CTAB (cetyltrimethylammonium bromide)
stabilized nanorods confirmed the characteristics of the
prepared material (Figure 3). Then, the nanorods were
treated with the thiol-functionalized epoxide EPT1 in
HEPES buffer (pH 7.2, 20 mm), followed by centrifugation
to separate the functionalized nanorods. After the removal of
any unreacted epoxide EPT1, the thermal decomposition of
nanorod-tethered EPT1 (EPT1–GNR) was studied. Quanti-
tative analysis of the spectroscopic data reveals that each
9
GNR carries about 6.5 ” 10 EPT1 molecules (see the
Supporting Information).
As the temperature of the DMSO solution containing the
singlet-oxygen trap DPFB and the EPT1–GNR conjugate was
increased, the DPBF peak around 414 nm decreased in
intensity. When the same solution was irradiated with
a laser at 830 nm at room temperature, a decrease in the
absorbance of the singlet-oxygen trap was observed, whereas
for GNRs alone, no changes were observed when irradiated in
the same manner (Figure 4).
Figure 3. TEM images of the CTAB–GNR conjugates (a,b) and the
EPT1–GNR conjugate (c). d) Normalized electronic absorption spectra
of CTAB–GNR and EPT1–GNR in HEPES (pH 7.2, 20 mm) and of
EPT1–GNR in DMSO.
Finally, we wanted to demonstrate the viability of the
GNR–endoperoxide agent as a remote-controlled source of
singlet oxygen in cell cultures. To that end, HeLa cells were
Figure 4. Decomposition and singlet-oxygen generation of plasmonically heated EPT1–GNR conjugates. Left: Decrease in the absorbance at
1
4
14 nm of DPBF in DMSO in the presence of EPT1–GNR upon heating in an oil bath. Right: Relative O generation efficiency of EPT1–GNR and
2
À2
CTAB–GNR only in DMSO, detected by the absorbance decrease of DPBF at 414 nm with time. Excitation was at 830 nm (1.0 Wcm ). During the
first 4 min, the samples were kept in the dark.
3
608 www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 3606 –3610

Angewandte
Communications
Chemie
cultures containing EPT1–GNR resulted in the fluorescence
labeling of the outer leaf of the cell membranes, an indication
of apoptosis. Again, the addition of azide resulted in a very
large suppression of cell death. MTT [3-(4,5-dimethylthiazol-
2
-yl)-2,5-diphenyltetrazolium bromide] colorimetric assays
(Figure 7) of metabolic activity provided a quantitative
picture supporting the same conclusion. A low concentration
of near-IR-irradiated EPT1–GNR provides singlet oxygen,
which could initiate apoptotic cell death in HeLa cell cultures.
Cell viability increases with the added azide, again demon-
strating the fact that cell death is due to the generation of
singlet oxygen.
Figure 5. ROS generation by EPT1–GNR in cell cultures. HeLa cells
were incubated with no additive (a), GNR–PEG (10 pM; b), EPT1–
GNR (10 pM; c), or EPT1–GNR (10 pM) and NaN (10 mm; d). All
3
À2
cells were irradiated with a 808 nm laser (2.0 Wcm , 10 min) and
ꢀ
incubated with an ROS sensor (Cyto-ID oxidative-stress detection kit)
for 30 min. The cell images were acquired by confocal microscopy.
Top: ROS sensor; middle: DAPI; bottom: merged with DIC.
The confocal microscopy results are highly revealing and
in accordance with our expectations (Figure 5). The control
compound GNR–PEG did not generate oxidative stress in the
Figure 7. MTT assays of cell viability. HeLa cells were incubated with
À2
1
0 pm of GNR–PEG or EPT1–GNR for 24 h, washed with DPBS, and
cell cultures when irradiated at a fluence rate of 2.0 Wcm
À2
irradiated with a 808 nm laser (2.0 Wcm , 10 min). After 24 h, the cell
viability was determined by an MTT test.
(
Figure 5). EPT1–GNR irradiation, however, caused fluores-
cence emission in the cytosol, lighting up the reactive oxygen
sensor. Inhibition by the added azide (Figure 5d) identified
the reactive oxygen species as singlet oxygen.
In a similar experiment, cells undergoing apoptosis were
identified with fluorescently labeled Annexin V (Figure 6).
Corroborating the ROS results, only the irradiation of cell
In conclusion, we have demonstrated that chemically
generated singlet oxygen can induce apoptosis and lead to the
death of cancer cells in cell cultures. The photodynamic
therapy of cancer, which rests on the success of the photo-
sensitized generation of singlet oxygen in tumors, has been
stunted by the problems of tumor hypoxia (both induced and
chronic) and light penetration, confining it to superficial
lesions. The chemical generation of singlet oxygen by the
controlled cycloreversion of endoperoxides has the potential
to alleviate these very critical problems. It is clear that even
a small quantity of singlet oxygen is capable of creating
a significant apoptotic response, most likely through amplifi-
cation mechanisms. Thus the fact that endoperoxides are
stoichiometric agents does not present a problem. A near-IR
light source and gold nanorods were used in this proof-of-
principle study, but the thermal cycloreversion of endoper-
oxides can also be remotely triggered by alternating magnetic
fields by using endoperoxides tethered to iron oxide nano-
particles.
We believe that more than one century after the first
reports on photodynamic therapy, its principal tenet is on the
verge of a radical change. The controlled chemical generation
of singlet oxygen may improve the application and availability
of endoperoxide-based therapies (EPT). Having its roots in
PDT, EPT may deliver all of the promises of PDT without its
inherent limitations. Work to realize these goals is in progress
in our laboratories.
Figure 6. Apoptosis induced by EPT1–GNR in cell cultures. HeLa cells
were incubated with no additives (a), GNR–PEG (10 pM; b), EPT1–
GNR (10 pM; c), or EPT1–GNR (10 pM) and NaN (10 mm; d). All
3
À2
cells were irradiated with a 808 nm laser (2.0 Wcm , 10 min),
incubated for 16 h, and stained with Annexin V and DAPI for 20 min.
The cell images were acquired by confocal microscopy. Top: Annexin V;
middle: DAPI; bottom: merged with DIC.
Angew. Chem. Int. Ed. 2016, 55, 3606 –3610
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 3609

Angewandte
Communications
Chemie
[
[
13] E. J. Kim, S. Bhuniya, H. Lee, H. M. Kim, C. Cheong, S. Maiti,
K. S. Hong, J. S. Kim, J. Am. Chem. Soc. 2014, 136, 13888 – 13894.
14] G. Kong, G. Anyarambhatla, W. P. Petros, R. D. Braun, O. M.
Colvin, D. Needham, M. W. Dewhirst, Cancer Res. 2000, 60,
Acknowledgements
E.U.A. gratefully acknowledges support from Bilkent Uni-
versity. J.Y. acknowledges a grant from the National Research
Foundation of Korea (NRF) funded by the Korean govern-
ment (MSIP, 2012R1A3A2048814). We are grateful to Bora
Bilgic for the graphic design.
6
950 – 6957.
[
15] N. Huebsch, C. J. Kearney, X. Zhao, J. Kim, C. A. Cezar, Z. Suo,
D. J. Mooney, Proc. Natl. Acad. Sci. USA 2014, 111, 9762 – 9767.
[16] S. Stolik, J. A. Delgado, A. PØrez, L. Anasagasti, J. Photochem.
Photobiol. B 2000, 57, 90 – 93.
[
[
[
[
[
17] R. Sullivan, C. H. Graham, Cancer Metastasis Rev. 2007, 26, 319 –
31.
18] J. A. Bertout, S. A. Patel, M. C. Simon, Nat. Rev. Cancer 2008, 8,
67 – 975.
19] T. M. Busch, S. M. Hahn, S. M. Evans, C. J. Koch, Cancer Res.
000, 60, 2636 – 2642.
20] J.-M. Aubry, C. Pierlot, J. Rigaudy, R. Schmidt, Acc. Chem. Res.
2003, 36, 668 – 675.
21] H. H. Wasserman, K. B. Wiberg, D. L. Larsen, J. Parr, J. Org.
Chem. 2005, 70, 105 – 109.
Keywords: endoperoxides · nanorods · photodynamic therapy ·
photosensitizers · singlet oxygen
3
9
How to cite: Angew. Chem. Int. Ed. 2016, 55, 3606–3610
Angew. Chem. 2016, 128, 3670–3674
2
[
1] P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti,
S. O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene, D.
Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B. C.
Wilson, J. Golab, CA Cancer J. Clin. 2011, 61, 250 – 281.
[22] C. Pierlot, S. Hajjam, C. Barthelemy, J.-M. Aubry, J. Photochem.
Photobiol. B 1996, 36, 31 – 39.
[23] J. J. Baumes, J. J. Gassensmith, J. Giblin, J.-J. Lee, A. G. White,
W. J. Culligan, W. M. Leevy, M. Kuno, B. D. Smith, Nat. Chem.
[
[
2] D. E. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003,
3, 380 – 387.
3] T. J. Dougherty, J. E. Kaufman, A. Goldfarb, K. R. Weishaupt,
D. Boyle, A. Mittleman, Cancer Res. 1978, 38, 2628 – 2635.
4] J. Moan, Q. Peng, Anticancer Res. 2003, 23, 591 – 600.
5] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J. Controlled
Release 2000, 65, 271 – 284.
2
010, 2, 1025 – 1030.
[
[
[24] S. Benz, S. Nçtzli, J. S. Siegel, D. Eberli, H. J. Jessen, J. Med.
Chem. 2013, 56, 10171 – 10182.
[25] X. Liu, H. Chen, X. Chen, Y. Alfadhl, J. Yu, D. Wen, Appl. Phys.
Rev. 2015, 2, 011103.
[
[
[
[
6] A. P. Castano, P. Mroz, M. R. Hamblin, Nat. Rev. Cancer 2006, 6,
[
26] N. W. S. Kam, M. OꢁConnel, J. A. Wisdom, H. Dai, Proc. Natl.
Acad. Sci. USA 2005, 102, 11600 – 11605.
27] B. Nikoobakht, M. A. El-Sayed, Chem. Mater. 2003, 15, 1957 –
1962.
5
35 – 545.
7] C. A. Morton, K. E. McKenna, L. E. Rhodes, Br. J. Dermatol.
008, 159, 1245 – 1266.
8] M. Bio, P. Rajaputra, G. Nkepang, Y. You, J. Med. Chem. 2014,
7, 3401 – 3409.
[
2
5
[28] K. Otsu, K. Sato, Y. Ikeda, H. Imai, Y. Nakagawa, Y. Ohba, J.
Fujii, Biochem. J. 2005, 389, 197 – 206.
[29] A. M. Asadirad, Z. Erno, N. R. Branda, Chem. Commun. 2013,
9] A. Shao, Y. Xie, S. Zhu, Z. Guo, S. Zhu, J. Guo, P Shi, T. D.
James, H. Tian, W. H. Zhu, Angew. Chem. Int. Ed. 2015, 54,
4
9, 5639 – 5641.
7
275 – 7280; Angew. Chem. 2015, 127, 7383 – 7388.
[
[
10] I. Simsek-Turan, F. Pir-Cakmak, D. C. Yildirim, R. Cetin-Atalay,
E. U. Akkaya, Chem. Eur. J. 2014, 20, 16088 – 16092.
11] S. Kolemen, M. Isik, G. M. Kim, D. Kim, H. Geng, M.
Buyuktemiz, T. Karatas, X.-F. Zhang, Y. Dede, J. Yoon, E. U.
Akkaya, Angew. Chem. Int. Ed. 2015, 54, 5340 – 5344; Angew.
Chem. 2015, 127, 5430 – 5434.
[30] J.-J. Lee, A. GonÅalves, B. A. Smith, R. Palumbo, A. G. White,
B. D. Smith, Aust. J. Chem. 2011, 64, 604 – 610.
[
31] S. Martins, J. P. S. Farinha, C. Baleizao, M. N. Berberan-Santos,
Chem. Commun. 2014, 50, 3317 – 3320.
Received: October 28, 2015
Revised: December 7, 2015
Published online: February 4, 2016
[
12] B. Wang, H. Yuan, Z. Liu, C. Nie, L. Liu, F. Lv, Y. Wang, S. Wang,
Adv. Mater. 2014, 26, 5986 – 5990.
3
610
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 3606 –3610