Photodynamic therapy via FRET following bioorthogonal click reaction in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2015.11.014

Longer wavelength light (650-800 nm) is desired to treat large tumors in photodynamic therapy (PDT). However, shorter wavelength light is needed in PDT for thin tumors, not to cause undesirable local side effects. We proposed a strategy for stepwise optical imaging and PDT using a bioorthogonal click chemistry and fluorescence resonance energy transfer (FRET). We prepared azidyl rhodamine (Rh-N₃, clickable FD) and cyclooctynyl phthalocyanine [Pc-(DIBAC), clickable PS], with which, here, we demonstrate that the non-catalytic click chemistry is rapid and efficient in cancer cells and FRET from a fluorescence dye (FD) to a photosensitizer (PS) is sufficient to generate enough singlet oxygen killing cancer cells by using shorter wavelength light.

Full text of DOI:10.1016/j.bmcl.2015.11.014

Bioorganic & Medicinal Chemistry Letters 26 (2016) 145–148
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Photodynamic therapy via FRET following bioorthogonal click
reaction in cancer cells
Moses Bio a, Pallavi Rajaputra a, Youngjae You a,b,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma, Oklahoma City, OK 73117, United States
b Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Longer wavelength light (650–800 nm) is desired to treat large tumors in photodynamic therapy (PDT).
However, shorter wavelength light is needed in PDT for thin tumors, not to cause undesirable local side
effects. We proposed a strategy for stepwise optical imaging and PDT using a bioorthogonal click
chemistry and fluorescence resonance energy transfer (FRET). We prepared azidyl rhodamine (Rh-N3,
clickable FD) and cyclooctynyl phthalocyanine [Pc-(DIBAC), clickable PS], with which, here, we
demonstrate that the non-catalytic click chemistry is rapid and efficient in cancer cells and FRET from
a fluorescence dye (FD) to a photosensitizer (PS) is sufficient to generate enough singlet oxygen killing
cancer cells by using shorter wavelength light.
Received 25 August 2015
Revised 4 November 2015
Accepted 5 November 2015
Available online 10 November 2015
Keywords:
Photodynamic therapy
Non-muscle invasive bladder cancer
Fluorescence resonance energy transfer
Bioorthogonal click reaction
Ó 2015 Elsevier Ltd. All rights reserved.
Photodynamic therapy (PDT) has much less side effects than
more established treatments like chemotherapy and radiation
therapy in treating local tumors. PDT is a unique treatment modal-
ity involving a photosensitizer (PS) that absorbs photons upon illu-
mination, converting the photonic energy to chemical energy.1–4
The photodynamic process generates cytotoxic singlet oxygen that
damages tumors. PDT was approved for the clinical treatment of
diseases including the wet form of age-related macular degenera-
tion, esophageal and lung cancers, and actinic keratosis.
Bladder cancer, in particular non-muscle invasive bladder can-
cer (NMIBC), was a prime target for PDT in the early PDT era due
to easy accessibility of light to bladder lumen through the urethral
track.5,6 However, PDT for NMIBC was not successful due to
bladder dysfunction after PDT.7 PDT with red light (630 nm) for
exciting Photofrin, although NMIBC resides in thin layers of urothe-
lium and lamina propria,8,9 damaged bladder muscle.10 For such
thin tumors, shorter wavelength light can be more beneficial in
minimizing damage of normal tissues under the tumors, which is
counterintuitive to the conventional PDT approach where longer
wavelength light is preferred for deeper tissue penetration. In the
bladder PDT, high specificity of PSs to tumors is also required to
minimize collateral damage to normal epithelium because a whole
bladder is illuminated to treat multifocal cancers.11
the fluorescent diagnosis of NMIBC in 2010.15,16 It is intravesically
administered for cancer cells to accumulate protoporphyrin IX.
Although it was demonstrated to improve detection sensitivity of
small tumors, further improvement is necessary. PpIX is not an
ideal fluorescence probe because it has low fluorescence quantum
yield as well as poor solubility in water causing aggregation and
fluorescence self-quenching.
To overcome such problems in detection and treatment of
NMIBC, we proposed a new strategy where the fluorescence imag-
ing and PDT are performed stepwise. First, tumors can be detected
by fluorescence imaging using fluorescence dye (FD). After a
bioorthogonal click reaction of FD and PS to form FD–PS conjugate
in cancers, singlet oxygen can be generated from FD–PS excited by
either shorter wavelength light hv1 or direct activation using
longer wavelength light hv3 of PS (Fig. 1). Use of shorter wave-
length light hv1 will minimize muscle damage in treating NMIBC.
There are three key questions to be addressed for this strategy to
be successful. (1) Can the FD be specifically delivered to cancer
cells using bladder cancer targeting vectors? (2) Can the click
reaction be accomplished in a short period time (ꢀ1 h) in cancer cells?
(3) Can singlet oxygen be generated from FD–PS enough via the FRET
to kill cancer cells after exciting FD with shorter wavelength light hv1?
In this Letter, we provide clear answers for keys questions # (2)
and # (3).
Recently, optical imaging has emerged as a very useful tool for
detecting small invisible cancers to improve surgical outcome.12–14
FDA approved HAL (hexyl-5-aminolevulinic acid) was approved for
We used azide-functionalized rhodamine (Rh-N3, clickable FD)
and cyclooctyne-functionalized phthalocyanine [Pc-(DIBAC), click-
able PS] for a bio-orthogonal copper-free click reaction.17–26 We
chose the combination of Rh and Pc because effective energy
⇑
Corresponding author. Tel.: +1 405 271 6593x47473.
http://dx.doi.org/10.1016/j.bmcl.2015.11.014
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

146
M. Bio et al. / Bioorg. Med. Chem. Lett. 26 (2016) 145–148
Step I: Fluorescence Imaging
hv1
Step II, Case 1: Selective PDT via FRET
hv1
A
0.8
0.6
0.4
0.2
0.0
hv1
hv2
Rh-N3
Pc-(DIBAC)
Rh-(DIBAC)-Pc
FRET
1O2
1O2
PS
PS
X
PS
FD
FD
bio-orthogonal
click reaction
3O2
3O2
Step II, Case 2: direct activation
hv3
1O2
PS
FD
400
500
600
700
3O2
Wavelength (nm)
B
Figure 1. Selective activation of FD–PS via FRET using hv1, avoiding activation of
non-conjugated PS or direct activation (using hv3) of PS. If FD is a FRET donor and PS
is an acceptor, wavelength of hv1 is shorter than wavelength of hv3.
1000
500
0
Rh-N3
Rh-(DIBAC)-Pc
transfer from Rh (a FRET donor) to Pc (a FRET acceptor) has been
demonstrated.27
The synthesis of Rh-N3 and Pc-(DIBAC) involved two facile and
high-yielding reactions for each compound (Scheme 1A). For Rh-
N3, the first step was azide formation using sodium azide, followed
by esterification of rhodamine B and 3-azidopropan-1-ol using DCC
and DMAP. Pc-(DIBAC) was synthesized by amide formation
between DBCO acid and compound 2. The purity of Rh-N3 and
Pc-(DIBAC) was confirmed by HPLC, >95% (Figs. S8 and S9).
We first confirmed the FRET from Rh to Pc in Rh–(DIBAC)-Pc by
observing a dramatic decrease of Rh fluorescence. The absorption
spectra of Rh–(DIBAC)-Pc showed the characteristic absorption
peaks of its individual components Pc-(DIBAC) and Rh-N3 (Fig. 2A).
There was no significant change in electronic absorption of Rh and
Pc chromophores in Rh–(DIBAC)-Pc at the ground state. However,
we observed a dramatic decrease of Rh fluorescence emission in
Rh–(DIBAC)-Pc compared with Rh-N3 at the equimolar concentra-
tion, most likely due to FRET from Rh to Pc (Figs. 1B and 2B).
550
600
650
700
Wavelength (nm)
Figure 2. (A) Absorption spectra of Rh-N3, Pc-(DIBAC) and Rh–(DIBAC)-Pc in
acetonitrile. (B) Fluorescence emission spectra of Rh-N3 and Rh–(DIBAC)-Pc in
acetonitrile at 525 nm excitation (both at 0.05 lM). Inset—UV–vis spectra of Rh-N3
and Rh–(DIBAC)-Pc at the same absorbance at the Rh peak.
We then monitored the progress of the click reaction of Rh-N3
and Pc-(DIBAC) in acetonitrile using fluorescence emission
changes; that is, examining the decrease in Rh emission due to
FRET. The fluorescence emission from the Rh group was highest
at the beginning of the reaction before the click reaction. However,
as the reaction between Rh-N3 and Pc-(DIBAC) proceeded, the flu-
orescence of Rh gradually decreased due to the FRET from Rh to the
Pc (Fig. 3A). [There was no photobleaching of Rh-N3 by excitation at
525 nm for fluorescence determination (Fig. S10). The Rh fluorescence
decrease was not due to photobleaching of Rh moiety.] After the
bioorthogonal click reaction, Rh–(DIBAC)-Pc was formed (yield,
90%, 1H NMR and high mass in Figs. S11 and S13). In contrast,
the fluorescence of Pc (Fig. 3B) remained unchanged, meaning
there was no FRET from Pc to Rh in the Rh–(DIBAC)-Pc, consistent
with a previous report.22
A
We were able to monitor the click reaction in the cultured cells
(T-24 cells, human bladder carcinoma cells). We observed rapidly
diminished Rh fluorescence in the cells after the addition of
Pc-(DIBAC), due to the click reaction. The emission of Rh of the cells
decreased in a time-dependent manner after the addition of Pc-
(DIBAC) into the cells having Rh-N3 (Fig. 4A). The reduction of Rh
fluorescence reached maximum in 60 min. Confocal imaging of
the cells provided further evidence of the click reaction in the cells
(Fig. 4B). Most of the Rh fluorescence was quenched after 50 min
without observable morphological change of the cells (Fig. 4Bb’).
Singlet oxygen generation of Rh–(DIBAC)-Pc via FRET was eval-
uated using DPBF (1,3-diphenylisobenzofurance).28 Light, Rh-N3, or
Pc-(DIBAC) alone did not produce any significant DPBF oxidation
by 531 nm laser illumination, exciting Rh moiety. Only Rh–
(DIBAC)-Pc produced rapid DPBF oxidation due to FRET from Rh
to Pc (Scheme 1B). After 14 min of illumination, the illuminated
Rh–(DIBAC)-Pc with DPBF showed significantly lower DPBF
absorption than the control (DPBF only; p <0.001, Fig. 5). The gen-
eration of singlet oxygen via FRET [Rh–(DIBAC)-Pc + hv (531 nm at
B
Scheme 1. (A) Reaction sequence and prepared donor fluorophore (Rh-N3) and
acceptor PS (Pc-(DIBAC)); (B) bioorthogonal click reaction process.

M. Bio et al. / Bioorg. Med. Chem. Lett. 26 (2016) 145–148
147
A
B 500
500
400
300
200
100
0
Excitation at 525 nm
0 min
Excitation at 615 nm
400
300
200
100
0
60 min
0 min
60 min
700
Wavelength (nm)
650
750
550
600
650
700
Wavelength (nm)
Figure 3. Progress of the bio-orthogonal click reaction monitored by decreased Rh emission due to FRET: fluorescence spectra upon excitation at 525 nm ((A) Rh emission)
and 615 nm ((B) Pc emission) in acetonitrile.
B
A
500
400
300
200
100
0
0 min
30 min
60 min
120 min
550
600
650
Wavelength (nm)
Figure 4. (A) Progress of the bio-orthogonal click reaction in T-24 cell; monitored using decreased Rh fluorescence due to FRET excitation at 525 nm. The cells were incubated
for click reaction and then lysed right before fluorescence determination. (B) Confocal fluorescence image of Rh group (excitation at 543 nm laser and emission filter:
550–620 nm). After 10 min incubation with Rh-N3, the media was removed and fresh media was added without Pc-(DIBAC) (a), or with Pc-(DIBAC) (b–d) are the
corresponding bright field images of (a) and (b). The confocal image was taken after 50 min.
Illumination Time (min)
0.5 mW/cm2)] was compared with direct excitation [Rh–(DIBAC)-
0
5
10
15
Pc + hv (690 nm at 0.5 mW/cm2)]. We observed a slightly lower,
but comparable, generation of singlet oxygen with FRET compared
with the direct excitation. The FRET process in Rh–(DIBAC)-Pc was
efficient to sufficiently generate singlet oxygen.
DBPF at 531 nm
Rh-N3 at 531 nm
0.00
-0.01
-0.02
-0.03
Pc-(DIBAC) at 531 nm
Lastly, to examine whether the rapid click reaction and FRET
can be made in the cells to kill cancer cells after exciting Rh moiety,
we determined the phototoxicity of the sequential treatment with
equimolar Rh-N3 and Pc-(DIBAC). Its dark toxicity and photo-
toxicity of each component [Rh-N3 or Pc-(DIBAC)] (Fig. 6) were
used as controls. While the sequential treatment did not show
any significant dark toxicity, it killed most of cells after illumina-
tion at 531 nm (>90% phototoxicity), even at 5 Â 10À7 M. In com-
parison, neither Rh-N3 nor Pc-(DIBAC) alone showed significant
phototoxicity at the same range of concentrations (p >0.05,
Rh-(DIBAC)-Pc at 531 nm
Rh-(DIBAC)-Pc at 690 nm
Figure 5. Relative rates of DPBF oxidation by singlet oxygen generated after
illumination by 531 nm diode laser at 0.5 mW/cm2. 0.5
M of compound was mixed
with 100 m of DPBF and the mixture was illuminated for 14 min. Absorbance
readings at 414 nm were taken every 2 min.
l
l

148
M. Bio et al. / Bioorg. Med. Chem. Lett. 26 (2016) 145–148
Supplementary data
Rh-N3 + Pc-(DIBAC) - hv
Rh-N3 + Pc-(DIBAC) + hv
Rh-N3 + hv
Pc-(DIBAC) + hv
150
100
50
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.11.
014.
References and notes
1. Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380.
2. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.;
Moan, J.; Peng, Q. J. Natl. Cancer Inst. 1998, 90, 889.
3. Dougherty, T. J. J. Clin. Laser Med. Surg. 2002, 20, 3.
4. Yano, S.; Hirohara, S.; Obata, M.; Hagiya, Y.; Ogura, S.-I.; Ikeda, A.; Kataoka, H.;
Tanaka, M.; Joh, T. J. Photochem. Photobiol., C 2011, 12, 46.
5. Kelly, J. F.; Snell, M. E.; Berenbaum, M. C. Br. J. Cancer 1975, 31, 237.
6. Prout, G. R., Jr.; Lin, C. W.; Benson, R., Jr.; Nseyo, U. O.; Daly, J. J.; Griffin, P. P.;
Kinsey, J.; Tian, M. E.; Lao, Y. H.; Mian, Y. Z., et al. N. Engl. J. Med. 1987, 317, 1251.
7. Yavari, N.; Andersson-Engels, S.; Segersten, U.; Malmstrom, P. U. Can. J. Urol.
2011, 18, 5778.
8. NCCN. In NCCN Clinical Practice Guidline in Oncology, Bladder Cancer; Version 2,
2015; MS5.
9. Birder, L.; Andersson, K. E. Physiol. Rev. 2013, 93, 653.
10. Harty, J. I.; Amin, M.; Wieman, T. J.; Tseng, M. T.; Ackerman, D.; Broghamer, W.
J. Urol. 1989, 141, 1341.
0
Concentration (M)
Figure 6. Dark toxicity and phototoxicity of the sequential treatment of equimolar
Rh-N3 and Pc-(DIBAC), and phototoxicity of Rh-N3 and Pc-(DIBAC): hv = 5.6 mW/
cm2 for 30 min, 531 nm diode laser [experimental details can be found in the SI:
Rh-N3 in the media was removed before addition of Pc-(DIBAC)]. Phototoxicity
represents relative phototoxicity of treated cells compared with untreated control
cells (no hv and no drug).
11. Star, W. M.; Marijnissen, H. P.; Jansen, H.; Keijzer, M.; van Gemert, M. J.
Photochem. Photobiol. 1987, 46, 619.
12. Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B.
W.; Hasan, T. Chem. Rev. 2010, 110, 2795.
13. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. Chem. Rev. 2010,
110, 2620.
14. Vahrmeijer, A. L.; Hutteman, M.; van der Vorst, J. R.; van de Velde, C. J.;
Frangioni, J. V. Nat. Rev. Clin. Oncol. 2013, 10, 507.
15. Witjes, J. A.; Redorta, J. P.; Jacqmin, D.; Sofras, F.; Malmstrom, P. U.; Riedl, C.;
Jocham, D.; Conti, G.; Montorsi, F.; Arentsen, H. C.; Zaak, D.; Mostafid, A. H.;
Babjuk, M. Eur. Urol. 2010, 57, 607.
16. Rink, M.; Babjuk, M.; Catto, J. W.; Jichlinski, P.; Shariat, S. F.; Stenzl, A.; Stepp,
H.; Zaak, D.; Witjes, J. A. Eur. Urol. 2013, 64, 624.
Fig. 6). The high phototoxicity of the sequential treatment of
Rh-N3 and Pc-(DIBAC) could be attributed to the singlet oxygen
generation through FRET following the formation of Rh–(DIBAC)-
Pc in the cells because Rh-N3 and Pc-(DIBAC) themselves cannot
produce singlet oxygen and phototoxicity at 531 nm excitation.
During the illumination, there could be
a small amount of
Rh–(DIBAC)-Pc outside the cells due to the diffusion of
Rh–(DIBAC)-Pc from the cells. However, its contribution to photo-
toxicity should be minor because singlet oxygen has a short
half-life (ꢀ40 ns) in aqueous medium and a very short diffusion
distance of between 20 and 200 nm.29–31
In conclusion, we provided important key data as a basis of our
new stepwise strategy for PDT with shorter wavelength light for
NMIBC. The click reaction in the cells is adequately rapid enough
to provide clicked Rh–(DIBAC)-Pc conjugate in 60 min. FRET from
Rh to Pc is sufficient to generate singlet oxygen enough to kill can-
cer cells. We are currently evaluating targeting vectors for bladder
cancers to be added to clickable FD to advance our strategy.
17. Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.;
Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793.
18. Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320,
664.
19. Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G. J. Angew. Chem., Intl. Ed. 2008, 47,
2253.
20. Lallana, E.; Fernandez-Megia, E.; Riguera, R. J. Am. Chem. Soc. 2009, 131, 5748.
21. Poloukhtine, A. A.; Mbua, N. E.; Wolfert, M. A.; Boons, G. J.; Popik, V. V. J. Am.
Chem. Soc. 2009, 131, 15769.
22. Bernardin, A.; Cazet, A.; Guyon, L.; Delannoy, P.; Vinet, F.; Bonnaffe, D.; Texier,
I. Bioconjugate Chem. 2010, 21, 583.
23. Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010, 132, 3688.
24. Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.; van Hest, J. C. M.;
van Delft, F. L. Chem. Commun. 2010, 97.
25. Sanders, B. C.; Friscourt, F.; Ledin, P. A.; Mbua, N. E.; Arumugam, S.; Guo, J.;
Boltje, T. J.; Popik, V. V.; Boons, G. J. J. Am. Chem. Soc. 2011, 133, 949.
26. Kuzmin, A.; Poloukhtine, A.; Wolfert, M. A.; Popik, V. V. Bioconjugate Chem.
2010, 21, 2076.
Acknowledgements
27. Zhao, Z.; Chan, P. S.; Li, H.; Wong, K. L.; Wong, R. N.; Mak, N. K.; Zhang, J.; Tam,
H. L.; Wong, W. Y.; Kwong, D. W.; Wong, W. K. Inorg. Chem. 2012, 51, 812.
28. Lissi, E. A.; Encinas, M. V.; Lemp, E.; Rubio, M. A. Chem. Rev. 1993, 93, 699.
29. Skovsen, E.; Snyder, J. W.; Lambert, J. D. C.; Ogilby, P. R. J. Phys. Chem. B 2005,
109, 8570.
Youngjae You, PhD was supported by a Research Scholar Grant
(RSG-13-102-01-CCE) from the American Cancer Society. We
appreciate the helpful discussions with Drs. Robert Hurst and Joel
Slaton at the College of Medicine, OUHSC and Dr. Sukyung Woo at
the College of Pharmacy, OUHSC.
30. Niedre, M.; Patterson, M. S.; Wilson, B. C. Photochem. Photobiol. 2002, 75, 382.
31. Moan, J. J. Photochem. Photobiol., B 1990, 6, 343.