Imidazole-modified porphyrin as a pH-responsive sensitizer for cancer photodynamic therapy

Article abstract of DOI:10.1039/c1cc13328d

5,10,15,20-Tetrakis(N-(2-(1H-imidazol-4-yl)ethyl)benzamide)porphyrin produced twice as many singlet oxygen (¹O₂) molecules at pH 5.0 (quantum yield 0.53 ± 0.01) than at pH 7.4, whereas the ¹O₂ quenching rate was reduced by a factor of 2.5 for a pH change from 7.4 to 5.0.

Full text of DOI:10.1039/c1cc13328d

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COMMUNICATION
Imidazole-modified porphyrin as a pH-responsive sensitizer for cancer
photodynamic therapyw
Xianchun Zhu, Wentong Lu, Yazhou Zhang, Aisha Reed, Brandon Newton, Zhen Fan,
Hongtao Yu, Paresh C. Ray and Ruomei Gao*
Received 4th June 2011, Accepted 8th August 2011
DOI: 10.1039/c1cc13328d
5
,10,15,20-Tetrakis(N-(2-(1H-imidazol-4-yl)ethyl)benzamide)-
However, upon the protonation of amines, the quenching of
1
porphyrin produced twice as many singlet oxygen ( O
2
) molecules
the chromophore by electron-transfer was precluded; thus the
1
photosensitized production of O
at pH 5.0 (quantum yield 0.53 Æ 0.01) than at pH 7.4, whereas
2
could ensue. Very recently,
1
the O
2
quenching rate was reduced by a factor of 2.5 for a pH
Lee and co-workers prepared a polysaccharide/drug conjugate
in which glycol chitosan was grafted with 3-diethylaminopropyl
7
isothiocyanate, chlorine e6 and poly(ethylene glycol). At
change from 7.4 to 5.0.
Photodynamic therapy (PDT) is based on the administration
of a tumor-localizing sensitizer and its activation by light
absorption. This technique has been developed for the treat-
higher pH chlorine e6 is deactivated via autoquenching.
However, in tumor acidic conditions, a polysaccharide/drug
conjugate undergoes conformational change into a uncoiled
1
structure for O
1
–4
1
ment of various malignancies, in which singlet oxygen ( O
2
)
2
production. In other studies, Ogilby’s group
1
plays a key role in light-induced cell death. The selectivity of
PDT, however, leaves much to be desired because normal cells
are also able to accumulate sensitizers, which leads to a
prolonged skin photosensitization. One strategy used to solve
reported a DNA sequence-controlled on/off switchable O
2
1
1
sensitizer. In this case, a sensitizer and a quencher were kept
in close contact in the ‘‘off state’’ by DNA-programmed
assembly but were separated to switch the sensitizer ‘‘on’’
through a process of competitive DNA hybridization. Zheng
et al. constructed a two-component system consisting of a
pyropheophorbide sensitizer tethered with a small peptide
this problem is to build certain triggers (e.g., pH, bioaffinity)
1
into conventional sensitizers for controllable
O
release,
2
–7
5
which has been the subject of many recent studies.
1
The utility of O
12
in PDT or organic synthesis is influenced
sequence to a carotenoid, an effective quencher to both triplet
13
2
1
by competitive processes that deactivate O
1
states and O
to ground state
2
.
The tether could be selectively cleaved in the
2
3
oxygen ( O
energy transfer. Quenching of O
in terms of photosensitizing applications. The vibrational
2
) via various mechanisms including vibrational
presence of caspase-3 protease to release pyropheophorbide
1
8
,9
1
1
for O
can be both good and bad
production. The regulation of O by the interaction of
2 2
2
14
a sensitizer with single-walled carbon nanotubes, TiO
nano-
2
1
15
16
deactivation of O
2
has been used in stereoselective control
9
of enecarbamate photooxidation. A sensitizer that produces
particles and calf thymus DNA was also demonstrated.
Herein, we reported a new system in which a pH-controlled
imidazole on/off switch was incorporated into 5,10,15,20-
tetrakis(N-(2-(1H-imidazol-4-yl)ethyl)benzamide) porphyrin
(TIEBAP). The four carboxylic acid groups of 5,10,15,20-
tetrakis(4-carboxyphenyl)porphyrin (TCPP) were converted
1
O at an acidic pH but is deactivated at physiological pH
2
would benefit from the therapeutic selectivity in cancer treat-
ment because the pH in growing malignant tumors tends to be
1
0
somewhat lower than that in surrounding normal tissue.
O’Shea’s group prepared a supramolecular agent containing
an amine functional group, in which the pH-based reversibility
into carboxylic amides via a typical amide coupling procedure
1
with histamine (see ESIw). Selective control of O
2
was achieved
1
5
1
of O
the pK
the adjacent amine quenched the excited sensitizer, hence
2
generation was observed. At pH values greater than
via efficient quenching of triplet states and/or O at alkaline
2
of amines, the intramolecular electron transfer from
but not acidic pH. The design and function of TIEBAP are
shown in Scheme 1. A special feature in this design is that the
imidazole moieties of cationic porphyrin TIEBAP were separated
from the porphyrin ring by ethylbenzamide chain spacers,
thereby preventing the delocalization of the positive charges
onto a porphyrin ring system through direct coupling that
b
1
2
preventing the energy transfer that led to the production of O .
Department of Chemistry and Biochemistry, Jackson State University,
Jackson MS 39211, USA. E-mail: ruomei.gao@jsums.edu;
Fax: +1 601 979 3674; Tel: +1 601 979 3719
w Electronic supplementary information (ESI) available: Materials
and instruments, the synthetic approach of TIEBAP, spectra of
1
17–19
could adversely affect the triplet and O
The imidazole heterocyclic ring contained two nitrogen
atoms with protonated pK values of 7.0 and 14.9 and was
2
yields.
a
ESI-MS, NMR, UV-visible and fluorescence, measurements of F
D
by histamine, effect of pH on
O intensity, TIEBAP stability tests and cell viability MTT
1
functionalized as the proton receptor in TIEBAP. The proto-
nation occurred just below physiological pH. Our results
indicate that this pH-responsive sensitizer possesses an ability
and quenching rate constant of
O
2
1
initial
assay. See DOI: 10.1039/c1cc13328d
2
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10311–10313 10311

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1
Table 1 Observed first-order solvent deactivation rate constants of O
2
À6
(
D
k
d
) upon irradiation of 7.0 Â 10 M TIEBAP at 532 nm in air-saturated
2
O solutions of different pH
pH
k
5.0
1.3 Â 10
6.5
2.2 Â 10
7.4
3.2 Â 10
À1
4
4
4
d
/s
1
increased as shown in Table 1. Clearly, the quenching of O
2
in
the reaction media containing TIEBAP could be neglected in
acidic solutions but not in alkaline solutions. The fast decay of
1
Scheme 1 Design and function of pH-responsive TIEBAP for
O
2
production.
1
4
À1
O
2
in weak alkaline solution (k
d
= 3.2 Â 10 s at pH 7.4)
could have resulted from the quenching by both aggregates
to efficiently kill tumor cells in an acidic environment upon
1
visible irradiation, whereas the production of O was drama-
and the imidazole moieties (see quenching results below).
1
Fig. 1 clearly shows how O generation was virtually switched
2
tically reduced at a physiological pH. We attributed this
2
on in response to decreasing pH.
1
therapeutic selectivity to the improved solubility of TIEBAP
1
The quantum yield of O production (F ) for TIEBAP at
and its inertness toward
O
2
in acidic solutions due to the
2
D
pH 5.1 D O was determined to be 0.53 Æ 0.01 by a relative
protonation of imidazole ring nitrogen. Sensitizer aggregation
occurred upon the deprotonation of imidazole ring nitrogen at
slightly alkaline pH, leading to an inefficient formation and
2
method in comparison to a well-developed reference sensitizer
meso-tetrasulfonatophenyl porphyrin (TSPP) with a known
2
6
1
FD 0.63 in D O (see ESIw). This value (0.53) is consistent
potential quenching of the triplet state and/or O
2
.
2
1
5
with the F
To quantify the effect of imidazole protonation on
D
of TCPP (0.53) in the weak alkaline solutions.
The positive charges at the imidazole moieties could impact
the charge distribution of the porphyrin ring only through
inductive effects. An increase in pH promoted the formation of
face-to-face H-type aggregation (see ESIw). The change in the
UV-visible spectrum of TIEBAP indicated dissociation of the
H-type aggregation of this porphyrin to the monomer upon
reducing the pH. In addition, the fluorescence spectra were
strongly affected by aggregation, showing a weaker two-band
emission spectrum at pH 8.2 but a strong one-band emission
spectrum at pH 5.3 (see ESIw). The aggregation is known to
reduce the quantum yield and lifetime of the excited triplet
states of porphyrins, thereby adversely affecting the quantum
1
O
2
quenching, we determined the total quenching rate constant
À1 À1
1
of O
2
removal (k , M
T
s ) by histamine in pH 7.4 and pH 5.1
D O solutions by the Stern–Volmer analysis. Measurements
2
were carried out at 532 nm excitation using TSPP as a sensitizer.
1
Our data indicated that the kinetics of O luminescence decay
2
at 1270 nm followed the Stern-Volmer equation (eqn (1)).
k
obs = k
d
+ k
T
[histamine]
(1)
1
where kobs is the observed 1st-order rate constant of O
1
2
decay
d 2
is the 1st-order rate constant of O decay
after laser pulse. k
in the absence of histamine. Changes in
1
20,21
1
yield of O
2
production (F
The effect of pH on O production was studied by monitoring
D
).
2
O lifetimes were
1
2
observed by the addition of histamine into the solutions
(Fig. 2). Stern–Volmer plots showed a good linear correlation
between kobs and quencher histamine concentrations, giving
1
the O luminescence at 1270 nm as previously described.
15,22
2
All of the experiments were performed in D O solutions with
2
1
different pH values, in which O had a longer lifetime (67
7
À1 À1
2
24
k (5.8 Æ 0.9) Â 10 M
s in pH 7.4 phosphate D O buffer
2
T
2
3
5
À1 À1
ms) compared to H
2
O (4 ms). Keeping the amount of
and (5.1 Æ 0.7) Â 10 M
s in pH 5.1 acetic acid/acetate
1
sensitizer constant, the intensity of
2
O was decreased with
D O buffer. Our results are comparable to literature values for
2
7
À1 À1
an increase in pH (Fig. 1 and ESIw). Compared to the initial
histidine, wherein k was determined to be 5.0 Â 10 M
s
T
1
value of O
1
6
À1 À1
27
at pH o pK . Except
2 2
intensity at pH 5.0, the production of O was
at pH 4 pK but less than 10 M
s
a
a
reduced by 30% at pH 6.5 and by 50% at pH 7.4, whereas
1
the lifetime of O in D O was increased by a factor of 2.2 at
2 2
T
for the physical quenching, the large k value at higher pH
1
could involve the chemical reactions of O
2
with the deproto-
2
nated imidazole rings via a [4 + 2] cycloaddition. Appar-
8
pH 5.0 (71 ms) and 1.5 at pH 6.5 (45 ms) compared to the
value of 31 ms at pH 7.4. The first-order solvent deactivation
1
ently, the efficient quenching of
1
could result in the limited utility of O2 in weak alkaline
2
O by imidazole moieties
1
4
À1
rate constants of O (k ) were measured to be 1.3 Â 10 s
2
d
at pH 5.0, which was consistent with the literature value of
25
.5 Â 10 s in D O. The observed k at higher pH values
4
À1
1
2
d
1
Fig. 2 Stern–Volmer plots for the luminescence quenching of
by TIEBAP in 0.1 M phosphate D
(black dots) and in 0.1 M acetic acid/acetate D
O
2
1
Fig. 1 Kinetics of
À6
O
2
decay after irradiation of air-saturated
2
O buffer solution of pH 7.4
O buffer solution of
7
.0 Â 10 M TIEBAP solutions (with 1% methanol in D
2
O) at
2
5
32 nm and at pH 5.0 (black), 6.5 (red) and 7.4 (blue); dots: experimental
pH 5.1 (red dots). Solid lines are theoretical simulation using a linear
least-square fitting method.
results and solid lines: first-order kinetic simulation
1
0312 Chem. Commun., 2011, 47, 10311–10313
This journal is c The Royal Society of Chemistry 2011

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system can be extended to other sensitizers modified with
imidazole groups.
We thank the support from National Science Foundation
(
NSF-PREM DMR-0611539). This work was also partially
supported by National Institutes of Health (NIH-RCMI
G12RR013459 and NIH-RTRN U54RR022762) and NSF
HRD-1008708. Any opinions, findings, and conclusions or
recommendations are those of the authors and do not reflect
the views of NSF or NIH.
Fig. 3 SK-BR-3 cancer cell viability after 10 minute (samples 2)
and 20 minute (samples 4) irradiation (at an average intensity of
À2
À5
6
7
.3 mW cm ) of 1.0 Â 10 M TIEBAP-attached 1 Â 10 breast
Notes and references
cancer cells at pH 7.4 (blue bars) and pH 6.1 (red bars). Samples 1 and
3
2
are set to 100% and used as references that represent 10 minute and
0 minute dark control in the absence of TIEBAP, respectively.
1
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rate constants of O
also extracted from the Stern–Volmer analysis as 1.4 Â 10 s
for both pH 5.1 and 7.4, which was consistent with the
2 2 d
decay in D O buffer solutions (k ) were
4
À1
4
À1
23
literature value of 1.5 Â 10 s for D
2
O.
5
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to acidic pH. For the same amount of sensitizer, the cytotoxicity
was significantly enhanced at pH 6.1 (red bars) when compared
to results at pH 7.4 (blue bars). When the pH decreased from
1
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.4 to 6.1, the cell viability was reduced from 95% to 75%
(
samples 2 in Fig. 3) and from 70% to 55% (samples 4 in
1
Fig. 3) after visible irradiation of TIEBAP for 10 and 20 minutes,
respectively. The cancer cells at pH 6.1 required about half of
the irradiation time to obtain the same cytotoxic effectiveness
compared to results at pH 7.4. The control experiments
performed in darkness at both pH 6.1 and pH 7.4 were used
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1
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that were separated from the porphyrin ring by ethylbenzamide
chain spacers to prevent the direct charge distribution onto the
porphyrin chromospheres. With an easy synthetic approach,
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rings in weak alkaline solutions makes imidazole moieties ideal
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2
7
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This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10311–10313 10313