A dual activatable photosensitizer toward targeted photodynamic therapy

Article abstract of DOI:10.1021/jm500456e

An unsymmetrical bisferrocenyl silicon(IV) phthalocyanine has been prepared in which the disulfide and hydrazone linkers can be cleaved by dithiothreitol and acid, respectively. The separation of the ferrocenyl quenchers and the phthalocyanine core greatly enhances the fluorescence emission, singlet oxygen production, intracellular fluorescence intensity, and in vitro photocytotoxicity. The results have been compared with those for the two symmetrical analogues which contain either the disulfide or hydrazone linker and therefore can only be activated by one of these stimuli. For the dual activatable agent, the greatest enhancement can be attained under a slightly acidic environment (pH = 4.5-6.8) and in the presence of dithiothreitol (in millimolar range), which can roughly mimic the acidic and reducing environment of tumor tissues. This compound can also be activated in tumor-bearing nude mice. It exhibits an increase in fluorescence intensity in the tumor over the first 10 h after intratumoral injection and can effectively inhibit the growth of tumor upon illumination.

Full text of DOI:10.1021/jm500456e

Article
pubs.acs.org/jmc
A Dual Activatable Photosensitizer toward Targeted Photodynamic
Therapy
Janet T. F. Lau, Pui-Chi Lo,* Xiong-Jie Jiang, Qiong Wang, and Dennis K. P. Ng*
Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
S
* Supporting Information
ABSTRACT: An unsymmetrical bisferrocenyl silicon(IV) phthalocya-
nine has been prepared in which the disulfide and hydrazone linkers can
be cleaved by dithiothreitol and acid, respectively. The separation of the
ferrocenyl quenchers and the phthalocyanine core greatly enhances the
fluorescence emission, singlet oxygen production, intracellular fluores-
cence intensity, and in vitro photocytotoxicity. The results have been
compared with those for the two symmetrical analogues which contain
either the disulfide or hydrazone linker and therefore can only be
activated by one of these stimuli. For the dual activatable agent, the
greatest enhancement can be attained under a slightly acidic environment
(pH = 4.5−6.8) and in the presence of dithiothreitol (in millimolar
range), which can roughly mimic the acidic and reducing environment of
tumor tissues. This compound can also be activated in tumor-bearing
nude mice. It exhibits an increase in fluorescence intensity in the tumor
over the first 10 h after intratumoral injection and can effectively inhibit the growth of tumor upon illumination.
called photodynamic molecular beacons,24−30 in which the
INTRODUCTION
■
photosensitizing unit is linked to a quencher via a cleavable
Photodynamic therapy (PDT) has emerged as a promising
peptide or a nucleic acid. In virtually all of these systems, their
treatment modality for some localized and superficial cancers,
photoactivity is triggered by a single stimulus. It is logical to
as well as certain noncancerous conditions.1,2 It involves the
extend the study to systems that can be activated by more than
combined action of a photosensitizer, light, and oxygen to
one tumor-associated stimulus. It is envisaged that these novel
generate cytotoxic reactive oxygen species, in which the
systems can exhibit further enhanced tumor selectivity. This
photosensitizer plays an extremely vital role in determining
strategy has been employed for intracellular delivery of
the therapeutic outcome. As a result, a large number of
anticancer drugs using pH- and redox-responsive car-
functional dyes3−6 and carriers7−10 have been studied with a
riers.31−33 However, to our knowledge, dual activatable
view to identifying desirable photosensitizing systems and
photosensitizers remain extremely rare. Ozlem and Akkaya
optimizing their photodynamic efficacy. To address the issue of
briefly reported a boron dipyrromethene based photosensitizer
selectivity toward malignant tissues, various approaches have
of which the singlet oxygen generation efficiency increased by
about 6-fold at low pH and high concentration of sodium ion in
been explored.11−14 These include bioconjugation to tumor-
specific vehicles such as the epidermal growth factor and
acetonitrile, but the study was not extended to biological
systems.34 As part of our continuing interest in pH- and redox-
monoclonal antibodies, and encapsulation in colloidal nano-
responsive photosensitizers,35−38 we report herein a novel
carriers such as polymeric micelles and silica-based nano-
particles. Recently, “smart” photosensitizing systems that can be
phthalocyanine-based photosensitizer that exhibits dual acid
activated selectively by tumor-associated stimuli have received
considerable attention.15,16 In these systems, the photo-
sensitizers are either self-quenched due to aggregation or
deactivated by the neighboring quenchers. Upon interactions
with the acidic and reducing environment of tumors, cancer-
related proteases, or mRNAs that have high tumor specificity,
the photosensitizers are disaggregated or detached from the
quenchers, resulting in restoration of their fluorescence and
photosensitizing properties. This approach can therefore
overcome the drawback of nonspecific activation of photo-
toxicity in PDT.
and thiol activation. Both stimuli are associated with the
characteristics of tumors39,40 and therefore are desirable targets
for activation.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Scheme 1 shows the
synthetic route for this dual activatable photosensitizer. We
employed ferrocenyl moieties as the quenchers,41 which were
linked to the phthalocyanine core via cleavable hydrazone and
Among the activatable photosensitizers reported so far, most
Received: December 27, 2013
Published: May 3, 2014
of them are self-quenched polymeric systems17−23 and the so-
© 2014 American Chemical Society
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Scheme 1. Synthesis of Dual Activatable Photosensitizer 4
Figure 1. Structures of phthalocyanines 5 and 6.
disulfide linkers. Silicon(IV) phthalocyanine dichloride (1) was
first treated with the less reactive alcohol 238 before the more
reactive benzylic alcohol 3 (Scheme S1 in Supporting
Information) was added. The triethylene glycol chain in 3
was introduced to enhance the solubility and reduce the
aggregation of the resulting phthalocyanine, both before and
after the cleavage. Under these conditions, the target
unsymmetrical phthalocyanine 4 was obtained in 15% yield,
while the symmetrical product 5 was isolated as the major
product (43% yield) and the other symmetrical phthalocyanine
638 was not obtained (Figure 1). The yield of 5 could be
increased to 79% by treating 1 with 3 only.
Compounds 4 and 5 were characterized with various
spectroscopic methods. The 1H NMR spectrum of 4 in
CDCl3 (Figure S1 in Supporting Information) clearly showed
all the expected signals. With reference to the spectra of 5 and 6
1
and with the aid of H−1H COSY spectroscopy (Figure S2 in
Supporting Information), all the signals could be assigned
unambiguously. It is worth mentioning that the protons that are
close to the silicon center are strongly shielded by the
phthalocyanine ring, resulting in a large upfield shift of their
signals (δ −1.77 and −0.38 for the oxyethylene protons of the
disulfide-linked ferrocene and δ −0.70 for the benzylic protons
of the hydrazone-linked ferrocene). In addition, as the two
ferrocenyl units of 4 are at different environment, they resonate
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as two sets of signals, each showing a singlet and two slightly
broadened signals with an integration ratio of 5:2:2, which are
typical for monosubstituted ferrocenes.
Electronic Absorption and Photophysical Properties.
These properties of 4−6 measured in N,N-dimethylformamide
(DMF) are listed in Table 1. The UV−vis spectrum of 4
the extent reached 62% after 6 h, 85% after 10 h, and 97% after
24 h. Compound 5, which does not have the disulfide linker,
showed negligible changes in fluorescence intensity up to 40
mM DTT.
The effect of pH on the fluorescence emission of 4−6 was
also examined. Figure S6 (Supporting Information) shows the
time-dependent fluorescence intensities of these compounds in
PBS at pH 4.5, 6.0, and 7.4, which were used to mimic the
tumor lysosomal compartment, tumor interstitial environment,
and extracellular environment of normal tissues, respectively.
For 5, there was no significant change in fluorescence intensity
over 12 h at pH 7.4, while the intensity increased significantly at
pH 6.0 and 4.5, particularly the latter. This observation could
be attributed to the hydrolytic cleavage of the hydrazone bonds
under an acidic environment, thus detaching the ferrocene-
based quenchers. The fluorescence intensity of 5 increased by
about 6-fold when the pH decreased from 7.4 to 4.5.
Compound 4 showed a moderate increase in fluorescence
intensity at pH 4.5 and 6.0 due to the release of only one
ferrocenyl moiety. For 6, which does not possess the acid-labile
hydrazone linkage, no significant fluorescence enhancement
was observed at pH 4.5−7.4.
Table 1. Electronic Absorption and Photophysical Data for
4−6 in DMF
a
b
c
compd
λmax (nm) (log ε)
λem (nm)
ΦF
ΦΔ
4
330 (4.84), 354 (4.86), 496 (3.47),
608 (4.53), 646 (4.49), 677 (5.29)
687
0.05
0.03
0.04
0.10
0.07
0.17
5
354 (4.79), 611 (4.48), 647 (4.43),
678 (5.25)
688
688
638
332 (4.90), 353 (4.86), 494 (3.67),
608 (4.51), 646 (4.44), 676 (5.29)
a
b
Excited at 610 nm. Using ZnPc in DMF as the reference (ΦF =
c
0.28). Using ZnPc as the reference (ΦΔ = 0.56 in DMF).
(Figure S3 in Supporting Information) showed typical
absorptions of metallophthalocyanines, together with two
additional bands at 330 and 496 nm assignable to the π-
conjugated ferrocenyl moieties.38 Its fluorescence quantum
yield (ΦF = 0.05) was much weaker than that of zinc(II)
phthalocyanine (ZnPc) (ΦF = 0.28) as a result of quenching by
the two ferrocenyl moieties. The singlet oxygen quantum yield
(ΦΔ) of 4 was also determined in DMF using 1,3-
diphenylisobenzofuran (DPBF) as the scavenger. The value
(ΦΔ = 0.10) was also significantly lower than that of ZnPc (ΦΔ
= 0.56). Among the three compounds, compound 5 showed
the lowest ΦF (0.03) and ΦΔ (0.07) values. This suggested that
the hydrazone-linked ferrocenyl moiety is a slightly stronger
quencher than the disulfide-linked counterpart, which may be a
result of its shorter separation from the phthalocyanine core.
The UV−vis spectrum of 4 in RPMI 1640 culture medium
with 0.5% Cremophor EL also showed a sharp and intense Q-
band, indicating that the compound remained nonaggregated
even in aqueous media. The fluorescence was also significantly
weaker than that of the reference compound containing two
axial -OCH2CH2S-SCH2CH2OH chains linked to the silicon-
(IV) phthalocyanine core. As expected, the fluorescence of 5
and 6 was also very weak because of the presence of the
ferrocenyl quenchers (Figure S4 in Supporting Information).
Redox- and pH-Responsive Properties. The redox-
responsive properties of 4 and 5 were first examined by
monitoring their changes in fluorescence intensity with time in
the presence of different concentrations of dithiothreitol
(DTT) in phosphate buffered saline (PBS). The results are
summarized in Figure S5 (Supporting Information), which also
includes the response of 6 reported earlier for comparison.38 It
can be seen that 6 shows a large increase in fluorescence
intensity in the presence of 10 or 40 mM DTT, while the
increase is small in the absence or presence of 2 μM DTT. With
reference that the intracellular glutathione concentration (1−10
mM) is significantly higher than the extracellular levels (2 μM
in plasma),42 the above conditions were used to mimic the
intracellular and extracellular reducing environments, respec-
tively. Fluorescence enhancement was also observed for 4, but
the effect was weaker because of the fact that there is only one
disulfide-linked ferrocenyl moiety in 4 and the noncleaved
hydrazone-linked ferrocenyl unit still partially quenches the
fluorescence. Nevertheless, the cleavage was confirmed and the
rate was monitored by HPLC. In the presence of 40 mM DTT,
The combined DTT and pH effect on the fluorescence
intensity of 4 is shown in Figure 2a. Higher intensity was
Figure 2. (a) Changes in fluorescence intensity of 4 (4 μM) with time
at pH 6.0, 6.8, or 7.4 and in the presence of 2 μM or 10 mM DTT in
PBS with 0.5% Cremophor EL. (b) Comparison of the rate of
photodegradation of DPBF sensitized by 4 under these conditions.
attained at higher DTT concentration (10 mM > 2 μM) and
lower pH (6.0 > 6.8 > 7.4). At 10 mM DTT and pH 6.0, the
extent of cleavage reached 28% after 6 h, 51% after 10 h, and
76% after 24 h as determined by HPLC analysis. It is clear that
the compound is responsive toward both stimuli. For 5 and 6,
their fluorescence intensity only responded to a single stimulus
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Figure 3. (a) Fluorescence images of MCF-7 cells after sequential incubation with DTT (0, 2 μM, or 4 mM) for 1 h, nigericin (25 μM) at pH 5.0 or
7.4 for 30 min, and 4 (1 μM) for 1 h. (b) Fluorescence images of MCF-7 cells after incubation with nigericin (25 μM) at pH 5.0, 6.0, or 7.4 for 30
min, followed by incubation with 5 (1 μM) for 1 h. The corresponding bright field images are shown in the upper row. (c) Comparison of the
relative intracellular fluorescence intensity of 4 at different DTT concentrations and pH values. Significant difference, compared with the intensity at
0 or 2 μM DTT at the same pH, is denoted by ∗, P < 0.001. (d) Comparison of the relative intracellular fluorescence intensity of 5 at different pH
values. Significant differences are denoted by ∗, P < 0.001, and #, P < 0.001, when compared with the intensity at pH 6.0 or 7.4 and with that at pH
5.0 or 7.4, respectively. Data are expressed as the mean standard deviation (number of cells, 30).
(either pH or DTT) as shown in Figure S7 (Supporting
Information).
In Vitro Studies. The DTT- and pH-dependent fluo-
rescence emission of 4 was also examined at the cellular level.
In this study, MCF-7 human breast cancer cells were
sequentially incubated with DTT (0, 2 μM, or 4 mM), the
ionophore nigericin which can equilibrate the intracellular and
extracellular pH (at pH 5.0 or 7.4),43 and 4, and then their
fluorescence images were captured and the intracellular
fluorescence intensities were determined (Figure 3a and Figure
3c). To better illustrate the extent of pH effect, the extremes of
a wider range (at pH 5.0 and 7.4) were used. At pH 7.4, very
weak fluorescence was observed in the absence or presence of 2
μM DTT. However, the fluorescence intensity increased by
about 3-fold when the DTT concentration was increased to 4
mM. This can be attributed to the cleavage of the disulfide
linker. At pH 5.0, the cells showed moderately strong
intracellular fluorescence in the absence or presence of 2 μM
DTT due to the cleavage of the acid-labile hydrazone linker.
Under these conditions, the intracellular fluorescence was
stronger than that at pH 7.4 and 4 mM DTT. This also
supported that the hydrazone-linked ferrocenyl moiety is a
stronger quencher than the disulfide-linked analogue as inferred
by comparing the fluorescence quantum yields of 5 and 6
(Table 1). The fluorescence intensity was particularly strong
when the cells were exposed to 4 mM DTT at pH 5.0, under
which both the disulfide and hydrazone linkages are cleaved,
rendering the compound to be fully “turned on”. For the
bis(hydrazone-linked) derivative 5, a similar increase in
intracellular fluorescence was also observed at lower pH. The
intensity increased by about 10-fold when the pH decreased
from 7.4 to 5.0 (Figure 3b and Figure 3d). The effect was
Apart from the fluorescence intensity, the effects of DTT and
pH on the singlet oxygen generation efficiency of these
compounds were also examined. Figure S8 (Supporting
Information) compares the rate of photodegradation of
DPBF sensitized by 4−6 upon exposure to 2 μM or 10 mM
DTT in PBS. All these compounds could not trigger the
formation of singlet oxygen in the dark (data not shown). Upon
illumination and at 2 μM DTT, all these compounds could
produce a small amount of singlet oxygen. In the presence of 10
mM DTT, the singlet oxygen generation efficiency increased
significantly and followed the order 5 < 4 < 6, which again
could be attributed to the different extent of disulfide cleavage.
Related studies were performed at different pH (4.5, 6.0, and
7.4). Similarly, under all these conditions, singlet oxygen could
not be generated in the dark (data not shown). Upon
illumination, the singlet oxygen generation efficiency followed
the trend 6 < 4 < 5, and for 4 and 5, the efficiency increased at
lower pH as shown in Figure S9 (Supporting Information)
reflecting the different extent of hydrazone cleavage.
Figure 2b shows the combined DTT and pH effect on the
singlet oxygen generation efficiency of 4. The results also
showed that 4 can be dual-activated. The highest efficiency was
attained at pH 6.0 and 10 mM DTT. When there was only one
favorable condition (i.e., either pH 6.0 or 10 mM DTT), the
efficiency was relatively lower but still higher than that under
the nonactivating condition (i.e., pH 7.4 and 2 μM DTT). As
shown in Figure S10 (Supporting Information), 5 and 6 were
only responsive to either pH (for 5) or DTT (for 6).
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stronger than that of 4, which only exhibited a 4-fold
enhancement. As the extracellular pH of tumors is slightly
acidic (6.4−6.8) while the normal tissues are generally
neutral,39 compound 5 is a potential fluorescent probe for
tumor.
The photodynamic activities of 4 and 5 were also evaluated
against MCF-7 cells. To reveal the effect of DTT on their
cytotoxicity, the cells were pretreated with different concen-
trations of DTT (0, 2 μM, or 4 mM) prior to incubation with
the phthalocyanines. Figure 4 shows the corresponding dose-
significantly when the DTT concentration is increased to 4
mM. The extent of decrease in IC50 value is slightly smaller
than that for 6, which can be attributed to the presence of one
additional disulfide-linked ferrocenyl moiety in 6. Compound 5
is also highly potent with IC50 values in the range of 73−76 nM,
which are not affected by the DTT concentration because the
compound does not contain DTT-cleavable disulfide linker.
Attempts to study the pH-dependent photocytotoxicity of these
compounds were not successful because most of the cells were
killed after being incubated with nigericin at pH 6.0 for 30 min
and left overnight for cell viability measurements.
The subcellular localization of 4 in MCF-7 cells, after the
treatment with DTT (4 mM), was further investigated by
confocal microscopy. As shown in Figure 5, the compound can
target the endoplasmic reticulum but not the mitochondria and
lysosomes of the cells. Similar results were observed for 6,38 but
they were contrary to those for our previously reported
silicon(IV) phthalocyanines which were localized in mitochon-
dria44−46 or lysosomes.35,47−50 Thus, the axial ligands play a
crucial role in controlling the subcellular localization of these
photosensitizers.
In Vivo Studies. Finally, the activation of 4 was
demonstrated in vivo. Nude mice bearing a HT29 human
colorectal carcinoma were treated with an intratumoral dose (1
μmol per kg body weight) of 4 or the noncleavable analogue of
6, in which the disulfide linker is replaced with an ethylene
group.38 Their whole-body fluorescence images were then
monitored and quantified continuously for 24 h (Figure 6).
The results clearly showed that the fluorescence of 4 was
greatly increased within the first 10 h, while that of the control
was negligible, showing that the fluorescence of 4 was restored
inside the tumor likely due to the cleavage of the linkers. In
addition, this compound could also effectively inhibit the
growth of tumor upon illumination as shown in Figure 7.
CONCLUSIONS
■
In summary, we have prepared and characterized two novel
silicon(IV) phthalocyanines. Compound 4 functions as a dual
pH- and redox-responsive photosensitizer. The fluorescence
intensity, singlet oxygen generation efficiency, intracellular
fluorescence, and in vitro photodynamic activity of this
compound are enhanced in a slightly acidic environment (pH
= 4.5−6.8) or in the presence of DTT (in millimolar range).
The greatest enhancement can be attained when both of these
two conditions occur, which can roughly mimic the acidic and
reducing environment of tumor tissues. The fluorescence and
photodynamic activity of this compound can also be activated
in vivo. Compound 5 shows an almost 10-fold increase in
intracellular fluorescence intensity when the pH drops from 7.4
to 5.0. Therefore, it also serves as a promising fluorescent probe
for tumor.
Figure 4. Cytotoxic effects of (a) 4 and (b) 5 on MCF-7 cells without
pretreatment with DTT (squares) or pretreated with 2 μM (circles) or
4 mM (triangles) DTT for 1 h prior to drug incubation for 6 h in the
absence (closed symbols) and presence (open symbols) of light (λ >
610 nm, 40 mW cm−2, 48 J cm−2). Data are expressed as the mean
standard error of the mean of three independent experiments, each
performed in quadruplicate.
dependent survival curves in the absence and presence of light.
Both compounds were noncytotoxic in the dark regardless of
the concentration of DTT but exhibited a high potency upon
illumination. The IC50 values are summarized in Table 2, which
also includes the data for 6 for comparison.38 It can be seen that
the photocytotoxicity of 4 remains nearly unchanged in the
absence and presence of 2 μM DTT but is enhanced
EXPERIMENTAL SECTION
■
Table 2. Half Maximal Inhibitory Concentration (IC50)
Values of 4−6 against MCF-7 Cells
a
General. All the reactions were performed under an atmosphere of
nitrogen. DMF was dried over barium oxide and distilled under
reduced pressure. Tetrahydrofuran (THF), toluene, and pyridine were
distilled from sodium benzophenone ketyl, sodium, and calcium
hydride, respectively. Chromatographic purifications were performed
on silica gel (Macherey-Nagel, 230−400 mesh) columns with the
indicated eluents. All other solvents and reagents were of reagent grade
and used as received. Compounds 2,38 7,51 10,52 14,38 and the
noncleavable analogue of 638 were prepared as described.
IC50 (nM)
compd
no DTT
2 μM DTT
4 mM DTT
4
100
75
2
105
76
6
64 1*
5
638
4
3
73
5
160
9
150
8
81 9*
a
Significant difference, compared with the data for no DTT or 2 μM
DTT, is denoted by ∗, P < 0.001.
1H and 13C{1H} NMR spectra were recorded on a Bruker
AVANCE III 400 spectrometer (1H, 400 MHz; 13C, 100.6 MHz) in
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Figure 5. Visualization of intracellular fluorescence for MCF-7 cells by using filter sets specific for 4 (in red, column 2) and ER-Tracker,
MitoTracker, or LysoTracker (in green, column 3). The corresponding bright field and merged images are shown in column 1 and column 4,
respectively.
CDCl3. Spectra were referenced internally by using the residual solvent
(1H, δ = 7.26) or solvent (13C, δ = 77.0) resonances relative to SiMe4.
Electrospray ionization (ESI) mass spectra were recorded on a
Thermo Finnigan MAT 95 XL mass spectrometer. UV−vis and
steady-state fluorescence spectra were taken on a Cary 5G UV−vis−
NIR spectrophotometer and a Hitachi F-7000 spectrofluorometer,
respectively. The fluorescence quantum yields (ΦF) of the samples (in
the inorganic salt. The crude product was then subject to column
chromatography using CHCl3 and then CHCl3/EtOH (50:1 v/v) as
the eluents. The product was isolated as a pale yellow oily liquid (1.21
g, 30%). Rf [CHCl3/EtOH (10:1 v/v)] = 0.32. 1H NMR (CDCl3): δ =
6.39 (s, 1 H, ArH), 6.33 (s, 1 H, ArH), 6.25 (s, 1 H, ArH), 4.48 (s, 2
H, CH2), 4.43 (s, 2 H, CH2), 4.21 (q, J = 7.2 Hz, 2 H, CO2CH2), 3.74
(s, 1 H, OH), 1.26 (t, J = 7.2 Hz, 3 H, CH3). 13C{1H} NMR (CDCl3):
δ = 169.6, 158.9, 157.4, 143.2, 107.6, 105.0, 101.5, 65.1, 64.6, 61.7,
14.0. MS (ESI): m/z 249 [M + Na]+ (100%). HRMS (ESI): m/z calcd
for C11H14NaO5 [M + Na]+, 249.0733; found, 249.0741.
DMF) were determined by the equation: ΦF(sample) = (Fsample
/
53
Fref)(Aref/Asample)(n2sample/n2ref)ΦF(ref)
,
where F, A, and n are the
measured fluorescence (area under the emission peak), the absorbance
at the excitation position (610 nm), and the refractive index of the
Preparation of 11. A mixture of 9 (0.80 g, 3.54 mmol), tosylate
10 (1.24 g, 3.89 mmol), and anhydrous K2CO3 (1.47 g, 10.64 mmol)
in acetone (20 mL) was heated under reflux overnight. The mixture
was then cooled to room temperature and filtered. The crude product
was subject to column chromatography using CHCl3 and then
CHCl3/EtOH (50:1 v/v) as the eluents. The product was isolated as a
colorless oily liquid (1.03 g, 78%). Rf [CHCl3/EtOH (50:1 v/v)] =
solvent, respectively. ZnPc in DMF was used as the reference [ΦF(ref)
=
0.28].54 The singlet oxygen quantum yields (ΦΔ) were measured in
DMF by the method of chemical quenching of DPBF by using ZnPc as
the reference (ΦΔ = 0.56).55
The purity of compounds 4 and 5 was determined by HPLC and
1H NMR spectroscopy, respectively, and was found to be ≥95%.
Reversed-phase analytical HPLC experiments were performed by
using an Apollo-C18 column (5 μm, 4.6 mm × 250 mm) and a Waters
Breeze 2 HPLC system with a 2998 photodiode array detector. The
conditions were set as follows: solvent A = 5% DMSO in acetonitrile;
solvent B = methanol. The gradient was 40% A + 60% B in the first 5
min, then changed to 100% A + 0% B in 25 min, kept under these
conditions for 10 min, and finally changed to 40% A + 60% B and
maintained under these conditions for a further 5 min. The flow rate
was fixed at 1.5 mL min−1.
Preparation of 9. A mixture of 3,5-dihydroxybenzyl alcohol (7)
(2.50 g, 17.84 mmol), ethyl bromoacetate (8) (2.98 g, 17.84 mmol),
and anhydrous K2CO3 (2.47 g, 17.87 mmol) in acetone (10 mL) was
heated under reflux for 6 h. The volatiles were evaporated under
reduced pressure. The residue was mixed with water, and then the pH
of the mixture was adjusted to ∼5 with 3 M HCl. The mixture was
then evaporated under reduced pressure at 80 °C. The residue was
redissolved in THF, and the resulting mixture was filtered to remove
1
0.29. H NMR (CDCl3): δ = 6.58 (s, 1 H, ArH), 6.51 (s, 1 H, ArH),
6.42 (s, 1 H, ArH), 4.61 (d, J = 6.0 Hz, 2 H, CH2), 4.59 (s, 2 H, CH2),
4.26 (q, J = 7.2 Hz, CO2CH2), 4.11 (t, J = 4.8 Hz, 2 H, CH2), 3.83 (t, J
= 4.8 Hz, 2 H, CH2), 3.71−3.73 (m, 2 H, CH2), 3.63−3.68 (m, 4 H,
CH2), 3.53−3.55 (m, 2 H, CH2), 3.37 (s, 3 H, OCH3), 1.30 (t, J = 7.2
Hz, 3 H, CH3). 13C{1H} NMR (CDCl3): δ = 168.8, 160.2, 159.1,
143.6, 106.3, 105.2, 101.1, 71.9, 70.8, 70.7, 70.5, 69.7, 67.6, 65.4, 65.1,
61.4, 59.0, 14.1. MS (ESI): m/z 395 [M + Na]+ (100%). HRMS
(ESI): m/z calcd for C18H28NaO8 [M + Na]+, 395.1676; found,
395.1676.
Preparation of 12. A mixture of hydrazine hydrate (80%, 2 mL)
and 11 (0.41 g, 1.10 mmol) in ethanol (2 mL) was stirred at room
temperature for 30 min. The mixture was then evaporated under
reduced pressure at 60 °C. The product was obtained as a colorless
oily liquid (0.34 g, 86%). 1H NMR (CDCl3): δ = 7.90 (br s, 1 H, NH),
6.57 (s, 1 H, ArH), 6.49 (s, 1 H, ArH), 6.35 (s, 1 H, ArH), 4.59 (s, 2
H, CH2), 4.48 (s, 2 H, CH2), 4.08 (t, J = 4.4 Hz, 2 H, CH2), 3.82 (t, J
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chromatography using CHCl3 and then CHCl3/MeOH (50:1 v/v)
as the eluents. The product was isolated as an orange oily liquid (0.45
g, 64%). Rf [CHCl3/MeOH (10:1 v/v)] = 0.29. 1H NMR (CDCl3): δ
= 9.27 (s, 1 H, CONH), 8.12 (s, 1 H, NCH), 6.63 (s, 1 H, ArH),
6.59 (s, 1 H, ArH), 6.46 (s, 1 H, ArH), 4.70 (s, 2 H, FcH), 4.65 (d, J =
5.6 Hz, 2 H, CH2), 4.59 (s, 2 H, CH2), 4.42 (s, 2 H, FcH), 4.21 (s, 5
H, FcH), 4.12−4.14 (m, 2 H, CH2), 3.84−3.86 (m, 2 H, CH2), 3.72−
3.74 (m, 2 H, CH2), 3.64−3.69 (m, 4 H, CH2), 3.54−3.56 (m, 2 H,
CH2), 3.38 (s, 3 H, CH3). 13C{1H} NMR (CDCl3): δ = 163.8, 160.2,
158.2, 151.4, 144.3, 106.5, 105.3, 101.0, 77.9, 71.9, 70.7, 70.6, 70.4,
69.6, 69.3, 69.2, 68.3, 67.7, 67.6, 64.7, 58.9. MS (ESI): m/z 555 [M +
H]+ (100%). HRMS (ESI): m/z calcd for C27H35FeN2O7 [M + H]+,
555.1788; found, 555.1784.
Preparation of 4. A mixture of silicon(IV) phthalocyanine
dichloride (1) (0.12 g, 0.20 mmol), alcohol 2 (0.13 g, 0.25 mmol),
and pyridine (1 mL, 12.36 mmol) in toluene (10 mL) was heated
under reflux for 2 h. To this reaction mixture, a solution of alcohol 3
(0.13 g, 0.23 mmol) and pyridine (0.5 mL, 6.18 mmol) in toluene (10
mL) was added, and the mixture was kept stirring under reflux for a
further 4 h. After evaporation of the volatiles in vacuo, the residue was
redissolved in CHCl3 to give a blackish green mixture, which was then
filtered through a bed of Celite to remove the insoluble black solid.
The greenish blue filtrate was evaporated, and the residue was
chromatographed using CHCl3 and then CHCl3/MeOH (50:1 v/v) as
the eluents. The two greenish blue bands were collected separately and
evaporated. The crude products were recrystallized from CHCl3/
hexane to afford 4 (48 mg, 15%) and 5 (0.14 g, 43%) both as a blue
1
solid. Rf [CHCl3/MeOH (10:1 v/v)] = 0.52. H NMR (CDCl3): δ =
9.61−9.63 (m, 8 H, Pc-Hα), 8.74 (s, 1 H, CONH), 8.33−8.35 (m, 8 H,
Pc-Hβ), 8.00 (s, 1 H, NCH), 7.91 (d, J = 8.8 Hz, 2 H, ArH), 7.73 (d,
J = 15.6 Hz, 1 H, CCH), 7.08 (d, J = 15.6 Hz, 1 H, CCH), 6.81
(d, J = 8.8 Hz, 2 H, ArH), 5.64 (s, 1 H, ArH), 4.73 (br s, 2 H, FcH),
4.58 (br s, 2 H, FcH), 4.47 (br s, 2 H, FcH), 4.45 (br s, 2 H, FcH),
4.39 (s, 2 H, CH2), 4.22 (s, 5 H, FcH), 4.16 (s, 5 H, FcH), 3.83 (s, 2
H, CH2), 3.56−3.63 (m, 8 H, CH2), 3.51−3.54 (m, 2 H, CH2), 3.41−
3.46 (m, 4 H, CH2), 3.36 (s, 3 H, CH3), 3.09 (virtual t, J = 4.8 Hz, 2
H, ArH), −0.38 (t, J = 6.0 Hz, 2 H, CH2), −0.70 (s, 2 H, CH2), −1.77
(t, J = 6.0 Hz, 2 H, CH2). The signal of one set of SCH2 protons was
overlapped with the water signal at δ 1.56. 13C{1H} NMR (CDCl3): δ
= 187.3, 167.3, 163.5, 159.7, 157.8, 156.1, 150.4, 149.4, 146.1, 141.7,
135.9, 133.5, 131.1, 130.5, 123.7, 118.8, 114.3, 102.2, 100.2, 79.3, 71.9,
71.3, 70.8, 70.7, 70.6, 70.5, 69.8, 69.3, 69.0, 68.4, 66.6, 66.5, 64.7, 62.5,
59.1, 53.8, 38.7, 35.8 (some of the signals were overlapped). MS (ESI):
m/z 1620 [M + H]+ (21%). HRMS (ESI): m/z calcd for
C84H75Fe2N10O12S2Si [M + H]+, 1620.3504; found, 1620.3507.
Preparation of 5. A mixture of silicon(IV) phthalocyanine
dichloride (1) (63 mg, 0.10 mmol), alcohol 3 (0.12 g, 0.22 mmol),
and pyridine (1 mL, 12.36 mmol) in toluene (15 mL) was heated
under reflux for 6 h. The volatiles were evaporated in vacuo. The
residue was redissolved in CHCl3 to give a blackish green mixture,
which was then filtered through a bed of Celite to remove the
insoluble black solid. The greenish blue filtrate was evaporated, and the
residue was chromatographed using CHCl3 and then CHCl3/MeOH
(10:1 v/v) as the eluents. The crude product was further purified by
size exclusion chromatography on Bio-Beads S-X1 beads (200−400
mesh) using THF as the eluent. It was then chromatographed again on
a silica gel column using CHCl3 and then CHCl3/MeOH (10:1 v/v)
as the eluents. The crude product was finally recrystallized from
CHCl3/hexane to afford a blue solid (0.13 g, 79%). Rf [CHCl3/MeOH
Figure 6. (a) Fluorescence images of tumor-bearing nude mice before
and after intratumoral injection of 4 or the noncleavable control (1
μmol per kg body weight) over 24 h. (b) Corresponding changes in
fluorescence intensity per unit area of the tumor. Five mice were used
for each compound.
Figure 7. Tumor growth delay after photodynamic treatment with 4.
Each of the five mice was treated with an intratumoral dose of 4 (1
μmol per kg body weight) followed by illumination with laser light at 7
and 48 h after injection (675 nm, 30 J cm−2). The mice for the control
(n = 4) were not treated with 4 and light. Data are expressed as the
mean standard derivation.
1
(10:1 v/v)] = 0.44. H NMR (CDCl3): δ = 9.59−9.61 (m, 8 H, Pc-
Hα), 8.75 (s, 2 H, CONH), 8.33−8.35 (m, 8 H, Pc-Hβ), 7.97 (s, 2 H,
NCH), 5.64 (s, 2 H, ArH), 4.72 (br s, 4 H, FcH), 4.44 (br s, 4 H,
FcH), 4.21 (s, 10 H, FcH), 3.81 (s, 4 H, CH2), 3.58−3.62 (m, 12 H,
CH2), 3.52−3.54 (m, 4 H, CH2), 3.42−3.45 (m, 8 H, CH2), 3.36 (s, 6
H, CH3), 3.06 (virtual t, J = 3.6 Hz, 4 H, ArH), −0.70 (s, 4 H, CH2).
13C{1H} NMR (CDCl3): δ = 163.2, 158.4, 156.2, 150.8, 149.4, 142.1,
135.9, 131.1, 130.9, 123.6, 102.2, 99.5, 71.9, 70.7, 70.6, 70.5, 69.3, 68.3,
66.5, 66.2, 59.0, 58.1 (some of the signals were overlapped). MS (ESI):
m/z 1648 [M + H]+ (5%). HRMS (ESI): m/z calcd for
C86H83Fe2N12O14Si [M + H]+, 1648.4643; found, 1648.4640.
= 4.4 Hz, 2 H, CH2), 3.70−3.72 (m, 2 H, CH2), 3.62−3.67 (m, 4 H,
CH2), 3.52−3.55 (m, 2 H, CH2), 3.36 (s, 3 H, CH3). 13C{1H} NMR
(CDCl3): δ = 168.4, 160.2, 158.2, 144.1, 106.3, 105.1, 100.8, 71.8,
70.7, 70.6, 70.5, 69.6, 67.5, 66.8, 64.7, 59.0. MS (ESI): m/z 359 [M +
H]+ (100%). HRMS (ESI): m/z calcd for C16H27N2O7 [M + H]+,
359.1813; found, 359.1813.
Preparation of 3. A mixture of ferrocenecarboxyaldehyde (13)
(0.32 g, 1.50 mmol) and 12 (0.45 g, 1.26 mmol) in ethanol (10 mL)
was heated under reflux for 2 h. The volatiles were evaporated under
reduced pressure. The crude product was subject to column
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Study of DTT- and pH-Responsive Fluorescence Emission.
Phthalocyanines 4−6 were dissolved in DMF to give 1 mM solutions,
which were diluted to 4 μM with PBS in the presence of 0.5%
Cremophor EL (v/v). DTT was dissolved in deionized water to give a
1 M solution. To study the effect of DTT on the fluorescence
intensity, a mixture of the phthalocyanine (4 μM) with DTT (2 μM,
10 mM, or 40 mM) or without DTT in PBS at pH 7.4 was prepared.
To examine the effect of pH on the fluorescence intensity, the study
was started readily after the phthalocyanine solution (1 mM in DMF)
was diluted to 4 μM with PBS at different pH (4.5, 6.0, or 7.4). To
study the combined effect of DTT and pH on the fluorescence
intensity, a mixture of the phthalocyanine (4 μM) with DTT (2 μM or
10 mM) in PBS at pH 6.0 or 7.4 was prepared. All these solutions were
stirred continuously at room temperature during the kinetic study, and
the fluorescence spectra (λex = 610 nm, λem = 630−800 nm) were
recorded at different time intervals.
Study of DTT- and pH-Responsive Singlet Oxygen Gen-
eration. DPBF was dissolved in DMF to give a 10 mM solution. To
study the effect of DTT on the singlet oxygen generation efficiency, a
mixture of phthalocyanine (4 μM) with DTT (2 μM or 10 mM) in
PBS at pH 7.4 was prepared. To examine the effect of pH, the study
was carried out after the phthalocyanine solution (1 mM in DMF) was
diluted to 4 μM with PBS at different pH (4.5, 6.0, or 7.4). To study
the combined effect of pH and DTT, a mixture of the phthalocyanine
(4 μM) with DTT (2 μM or 10 mM) in PBS at pH 6.0 or 7.4 was
prepared. The above phthalocyanine solutions were stirred continu-
ously at room temperature for 8 h. These solutions (3 mL) were then
mixed with the DPBF solution (10 mM, 21 μL), followed by
illumination with red light coming from a 100 W halogen lamp after
passing through a water tank for cooling and a color glass filter
(Newport, cut-on at 610 nm). The decay of DPBF at 411 nm was
monitored with time.
Study of Photocytotoxicity. DTT was dissolved in deionized
water to give a 1 M stock solution, which was diluted with the culture
medium to give 2 μM and 4 mM DTT solutions. Approximately 3 ×
104 cells per well in the culture medium were inoculated in 96-
multiwell plates and incubated overnight at 37 °C in a humidified 5%
CO2 atmosphere. The cells were rinsed with PBS and incubated with
DTT solution (2 μM or 4 mM, 100 μL) or the culture medium
(without DTT) (100 μL) for 1 h under the same conditions. The cells,
after being rinsed with PBS twice, were incubated with 100 μL of the
above drug solutions for 6 h at 37 °C under 5% CO2. The cells were
then rinsed again with PBS and refilled with 100 μL of the culture
medium before being illuminated at ambient temperature. The light
source consisted of a 300 W halogen lamp, a water tank for cooling,
and a color glass filter (Newport) cut-on 610 nm. The fluence rate (λ
> 610 nm) was 40 mW cm−2. Illumination of 20 min led to a total
fluence of 48 J cm−2.
Cell viability was determined by means of the colorimetric MTT
assay.56 After illumination, the cells were incubated at 37 °C under 5%
CO2 overnight. An MTT (Sigma) solution in PBS (3 mg mL−1, 50
μL) was added to each well followed by incubation for 2 h under the
same environment. A solution of sodium dodecyl sulfate (Sigma, 10%
by weight, 50 μL) was then added to each well. The plate was
incubated in an oven at 60 °C for 30 min, and then 80 μL of
isopropanol was added to each well. The plate was agitated on a Bio-
Rad microplate reader at ambient temperature for 10 s before the
absorbance at 540 nm for each well was taken. The average absorbance
of the blank wells, which did not contain the cells, was subtracted from
the readings of the other wells. The cell viability was then determined
by the following equation: % viability = [(Ai/Acontrol) × 100]/n, where
Ai is the absorbance of the ith data (i = 1, 2, ..., n), Acontrol is the average
absorbance of the control wells in which the phthalocyanine was
absent, and n (= 4) is the number of the data points.
Cell Lines and Culture Conditions. The MCF-7 human breast
cancer cells (ATCC, no. HTB-22) were maintained in RPMI 1640
medium (Invitrogen, no. 223400-21) supplemented with fetal calf
serum (10%), sodium pyruvate (1 mM), and penicillin−streptomycin
(100 units mL−1 and 100 μg mL−1, respectively). The HT29 human
colorectal carcinoma cells (ATCC, no. HTB-38) were maintained in
Dulbecco’s modified Eagle medium (Invitrogen, no.10313-021)
supplemented with fetal calf serum (10%), penicillin−streptomycin
(100 units mL−1 and 100 μg mL−1, respectively), L-glutamine (2 mM),
and transferrin (10 μg mL−1). Approximately 3 × 104 cells per well in
the media were inoculated in 96-multiwell plates and incubated
overnight at 37 °C in a humidified 5% CO2 atmosphere.
Study of DTT- and pH-Responsive Intracellular Fluores-
cence. Phthalocyanines 4 and 5 were first dissolved in DMF to give
1.6 mM solutions, which were diluted to 80 μM with the culture
medium in the presence of 0.5% Cremophor EL (v/v). These
solutions were then further diluted with the culture medium for the
following studies.
Subcellular Localization Studies. Approximately 6 × 104 MCF-7
cells in RPMI 1640 medium (2 mL) were seeded on a coverslip and
incubated overnight at 37 °C with 5% CO2. After removal of the
medium, the cells were first incubated with DTT in PBS (4 mM, 2
mL) for 1 h. They were then rinsed with PBS and incubated with a
solution of 4 in the medium (2 μM, 2 mL) for a further 2 h under the
same conditions. The medium was removed, and the cells were
incubated with ER-Tracker Green (Molecular Probes, 0.2 μM in PBS),
MitoTracker Green FM (Molecular Probes, 0.25 μM in the medium),
or LysoTracker Green DND 26 (Molecular Probes, 2.0 μM in the
medium) for a further 20 min. For all the cases, the cells were then
rinsed with PBS and viewed with an Olympus FV1000 IX81-SIM
confocal microscope equipped with a 488 nm multi-argon laser and a
635 nm diode laser. All the trackers were excited at 488 nm and
monitored at 510−560 nm, while 4 was excited at 635 nm and
monitored at 650−750 nm. The images were digitized and analyzed
using the FV10-ASW software. The subcellular localization of 4 was
revealed by comparing the intracellular fluorescence images caused by
the ER-Tracker, MitoTracker, or LysoTracker and 4.
In Vivo Imaging. Female Balb/c nude mice (20−25 g) were
obtained from the Laboratory Animal Services Centre at The Chinese
University of Hong Kong. All animal experiments had been approved
by the Animal Experimentation Ethics Committee of the University.
The mice were kept under pathogen-free conditions with free access to
food and water. HT29 cells (1 × 107 cells in 200 μL) were inoculated
subcutaneously at the back of the mice. Once the tumors had grown to
a size of 60−100 mm3, the mice were fed with low fluorescence diet
(TestDiet, no. AIN-93M) for 4 days. Phthalocyanine 4 or the
noncleavable analogue of 6, in which the disulfide linker is replaced
with an ethylene group (200 nmol), was first dissolved in
dimethylsulfoxide (15 μL) and Tween 80 (1 μL). The solutions
were then diluted to 1 nmol μL−1 with distilled water (184 μL). These
phthalocyanine solutions (25 μL) were injected intratumorally to the
tumor-bearing mice. In vivo fluorescence imaging was performed
before and after the injection (at different time points) with an
Odyssey infrared imaging system (excitation wavelength of 680 nm,
emission wavelength of ≥700 nm). The images were digitized and
About 6 × 104 MCF-7 cells were seeded on a coverslip and
incubated overnight at 37 °C under 5% CO2. The medium was
removed and rinsed with PBS. For the study of 4, the cells were first
incubated with DTT (2 μM or 4 mM) or without DTT in PBS (2 mL)
for 1 h under the same conditions. The cells were rinsed with PBS and
then incubated with nigericin (Sigma) in PBS (25 μM, 2 mL) at
different pH (5.0 or 7.4) for 30 min. The cells were then rinsed with
PBS and incubated with a solution of 4 in the medium (1 μM, 2 mL)
for 1 h. For the study of 5, the cells were incubated with nigericin in
PBS (25 μM, 2 mL) at different pH (5.0, 6.0, or 7.4) for 30 min. They
were then rinsed with PBS, followed by incubation with a solution of 5
in the medium (1 μM, 2 mL) for 1 h. The cells were then rinsed with
PBS twice and then viewed with a Leica SP5 confocal laser scanning
microscope equipped with a 633 nm helium−neon laser. The emission
signals from 650 to 760 nm were collected, and the images were
digitized and analyzed by Leica Application Suite Advanced
Fluorescence software. The average intracellular fluorescence
intensities (for a total of 30 cells in each sample) were also
determined.
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analyzed by the Odyssey imaging system software (no. 9201-500).
Five mice were used for each compound.
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In Vivo Photodynamic Treatment. The length, width, and
thickness of the tumors were measured by a micrometer digital caliper
(SCITOP Systems). The tumor volume (mm3) was calculated using
the following formula: tumor volume = π(length × width ×
thickness)/6. Once the tumors had grown to a size of 80−100 mm3,
the mice were injected intratumorally with a solution of 4 prepared as
described above (1 nmol μL−1, 25 μL). At 7 h after injection, the
tumor was illuminated with a diode laser (Biolitec Ceralas) at 675 nm
operating at 0.1 W. Illumination on a spot size of 1.0 cm2 for 5 min led
to a total fluence of 30 J cm−2. The illumination was repeated at 48 h
after injection. The tumor sizes of the nude mice were monitored
periodically for a duration of 10 d. The tumor volumes were compared
with a control group of mice without the drug and light treatment.
Five mice were used for the photodynamic treatment with 4, while
four mice were used for the control.
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Statistical Analyses. Data were analyzed with unpaired t test
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic scheme for 3, 1H NMR and 1H−1H COSY spectra of
4 in CDCl3, UV−vis spectra of 4 in DMF or RPMI 1640
culture medium, fluorescence spectra of 4−6 and 14 in the
culture medium, time-dependent changes in fluorescecne
intensity of 4−6 in the presence of different concentrations
of DTT and/or at different pH in PBS, and comparison of the
rate of photodegradation of DPBF sensitized by 4−6 upon
exposure to different concentrations of DTT and/or at different
pH in PBS. This material is available free of charge via the
Internet at http://pubs.acs.org.
(12) Schmitt, F.; Juillerat-Jeanneret, L. Drug targeting strategies for
photodynamic therapy. Anti-Cancer Agents Med. Chem. 2012, 12, 500−
525.
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Hamblin, M. R. Photodynamic therapy: photosensitizer targeting and
delivery. In Drug Delivery in Oncology: From Basic Research to Cancer
Therapy; Kratz, F., Senter, P., Steinhagen, H., Eds.; Wiley-VCH:
Weinheim, Germany, 2012; pp 1569−1603.
(14) Lim, C.-K.; Heo, J.; Shin, S.; Jeong, K.; Seo, Y. H.; Jang, W.-D.;
Park, C. R.; Park, S. Y.; Kim, S.; Kwon, I. C. Nanophotosensitizers
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AUTHOR INFORMATION
Corresponding Authors
*P.-C.L.: phone, +852-3943-1326; fax, +852-2603-5057; e-mail,
pclo@cuhk.edu.hk.
■
(16) Verhille, M.; Couleaud, P.; Vanderess, R.; Brault, D.; Barberi-
Heyob, M.; Frochot, C. Modulation of photosensitization processes
for an improved targeted photodynamic therapy. Curr. Med. Chem.
2010, 17, 3925−3943.
*D.K.P.N.: phone, +852-3943-6375; fax, +852-2603-5057; e-
mail, dkpn@cuhk.edu.hk.
Notes
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of novel protease-mediated photodynamic agent. Cancer Res. 2006, 66,
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(18) Gabriel, D.; Campo, M. A.; Gurny, R.; Lange, N. Tailoring
protease-sensitive photodynamic agents to specific disease-associated
enzymes. Bioconjugate Chem. 2007, 18, 1070−1077.
We thank Prof. W.-P. Fong and P.-W. Choi for help in the
confocal microscopic study. This work was supported by a
grant from the Research Grants Council of the Hong Kong
Special Administrative Region (Project No. 402211).
(19) Bae, B.-c.; Na, K. Self-quenching polysaccharide-based nanogels
of pullulan/folate-photosensitizer conjugates for photodynamic
therapy. Biomaterials 2010, 31, 6325−6335.
(20) Koo, H.; Lee, H.; Lee, S.; Min, K. H.; Kim, M. S.; Lee, D. S.;
Choi, Y.; Kwon, I. C.; Kim, K.; Jeong, S. Y. In vivo tumor diagnosis
and photodynamic therapy via tumoral pH-responsive polymeric
micelles. Chem. Commun. 2010, 46, 5668−5670.
(21) Park, S. Y.; Baik, H. J.; Oh, Y. T.; Oh, K. T.; Youn, Y. S.; Lee, E.
S. A smart polysaccharide/drug conjugate for photodynamic therapy.
Angew. Chem., Int. Ed. 2011, 50, 1644−1647.
ABBREVIATIONS USED
■
DMF, N,N-dimethylformamide; DPBF, 1,3-diphenylisobenzo-
furan; DTT, dithiothreitol; ESI, electrospray ionization; PBS,
phosphate buffered saline; PDT, photodynamic therapy; THF,
tetrahydrofuran; ZnPc, zinc(II) phthalocyanine; ΦF, fluores-
cence quantum yield; ΦΔ, singlet oxygen quantum yield
(22) Lee, S. J.; Koo, H.; Lee, D.-E.; Min, S.; Lee, S.; Chen, X.; Choi,
Y.; Leary, J. F.; Park, K.; Jeong, S. Y.; Kwon, I. C.; Kim, K.; Choi, K.
Tumor-homing photosensitizer-conjugated glycol chitosan nano-
particles for synchronous photodynamic imaging and therapy based
on cellular on/off system. Biomaterials 2011, 32, 4021−4029.
(23) Kim, H.; Mun, S.; Choi, Y. Photosensitizer-conjugated
polymeric nanoparticles for redox-responsive fluorescence imaging
and photodynamic therapy. J. Mater. Chem. B 2013, 1, 429−431.
(24) Zheng, G.; Chen, J.; Stefflova, K.; Jarvi, M.; Li, H.; Wilson, B. C.
Photodynamic molecular beacon as an activatable photosensitizer
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