A disulfide-linked conjugate of ferrocenyl chalcone and silicon(iv) phthalocyanine as an activatable photosensitiser

Article abstract of DOI:10.1039/c2cc37251g

A novel bis(ferrocenyl chalcone) silicon(iv) phthalocyanine has been prepared in which the disulfide linker can be cleaved by dithiothreitol. The separation of the ferrocenyl quencher and the phthalocyanine core greatly enhances the fluorescence emission,

Full text of DOI:10.1039/c2cc37251g

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A disulfide-linked conjugate of ferrocenyl chalcone and silicon(IV)
phthalocyanine as an activatable photosensitiserwz
Janet T. F. Lau, Xiong-Jie Jiang, Dennis K. P. Ng and Pui-Chi Lo*
Received 4th October 2012, Accepted 24th October 2012
DOI: 10.1039/c2cc37251g
A novel bis(ferrocenyl chalcone) silicon(IV) phthalocyanine has
been prepared in which the disulfide linker can be cleaved by
dithiothreitol. The separation of the ferrocenyl quencher and the
phthalocyanine core greatly enhances the fluorescence emission,
singlet oxygen production and in vitro photocytotoxicity.
probes for thiols11 and protein transduction12 in living cells. The
synthesis of this novel compound and the effects of a reducing
stimulus, namely dithiothreitol (DTT), on its photophysical
properties and in vitro photocytotoxicity are described below.
Scheme 1 shows the preparation of this compound and a
non-cleavable counterpart. Treatment of 3-ferrocenyl-1-(40-
hydroxyphenyl)-prop-2-en-1-one (1)9a with ethyl bromoacetate
in the presence of K2CO3 in acetone afforded compound 2,
which was hydrolysed with NaOH to give compound 3.
This compound was then coupled with 2-hydroxyethyl disulfide
(4a) or 1,6-hexanediol (4b) in the presence of N,N0-dicyclohexyl-
carbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP) and
1-hydroxybenzotriazole (HOBt) to give the corresponding
alcohols 5a and 5b. Further substitution of silicon(IV) phthalo-
cyanine dichloride (SiPcCl2) with these alcohols resulted in the
formation of the cleavable phthalocyanine 6a and the non-cleavable
analogue 6b. In addition, SiPcCl2 was also treated with an excess
of alcohol 4a to give SiPc(OCH2CH2S–SCH2CH2OH)2 (7) as a
reference compound, in which the two ferrocenyl chalcone moieties
are absent (Scheme S1, ESIz). The experimental details and the
characterisation data are given in ESI.z
There has been considerable interest in development of efficient
photosensitisers for photodynamic therapy (PDT).1 Apart from
their efficiency in generating reactive oxygen species (ROS), their
selectivity towards malignant tissues is also a critical factor
affecting the therapeutic outcome. To achieve the latter, various
strategies have been explored which include conjugation to
tumour-specific vehicles such as epidermal growth factor and
monoclonal antibody, and encapsulation in nano-systems such as
liposomes, polymeric micelles and silica-based nanoparticles.2 In
addition, activatable photosensitisers are also of much current
interest.2a,3 Upon interactions with various tumour-associated
stimuli such as the acidic environment of tumours,4 cancer-related
proteases,5 and mRNAs that have high tumour specificity,6 the
photosensitising ability of these photosensitisers can be triggered,
thereby offering control over their tumour selectivity. We have
recently reported a ketyl-linked self-quenched phthalocyanine
dimer as a rare example of activatable phthalocyanine-based
photosensitisers.7 Under an acidic environment (pH = 5.0–6.5),
the ketal linker is cleaved and the disconnection of the phthalo-
cyanine units enhances the photosensitising efficiency. We
describe herein another example which has a different mode of
activation. It contains a silicon(IV) phthalocyanine core linked to
two ferrocenyl chalcone moieties at the axial positions via a
disulfide linker. The ferrocenyl unit serves as a quencher for the
excited phthalocyanine8 and a potential chemotherapeutic agent,9
which may promote the anti-tumour effect after cleavage. The
incorporation of the disulfide linker, which can be cleaved by
biological reducing agents, provides a way for activation. In fact,
a substantial number of disulfide-linked systems have been prepared
and used for drug delivery and release,10 and as fluorescent
Department of Chemistry, The Chinese University of Hong Kong,
Shatin, N.T., Hong Kong. E-mail: pclo@cuhk.edu.hk;
Fax: +852 2603 5057
w This article is part of the ChemComm ‘Emerging Investigators 2013’
themed issue.
z Electronic supplementary information (ESI) available: Experimental
details, electronic absorption and photophysical data, cytotoxicity and
confocal microscopic images. See DOI: 10.1039/c2cc37251g
Scheme 1 Synthesis of ferrocenyl phthalocyanines 6a and 6b.
c
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The electronic absorption spectra of 6a, 6b and 7 in DMF
(Fig. S1a–c, ESIz) were typical for non-aggregated phthalo-
cyanines. They showed a B-band at 353–355 nm, an intense and
sharp Q-band at 674–676 nm together with two vibronic bands at
608 and 642–646 nm (Table S1, ESIz). The spectra of 6a and 6b
showed two additional bands at 330–332 and 494–498 nm, which
can be attributed to the absorptions of the ferrocenyl chalcone
moieties. It was supported by the absorption spectrum of 5a, which
displayed two absorption bands at 311 and 489 nm in DMF
(Fig. S1d, ESIz). Upon excitation at 610 nm, these phthalocyanines
showed a fluorescence emission at 684–688 nm. The fluorescence
quantum yields of 6a and 6b (FF = 0.04–0.05) were greatly
reduced compared with that of 7 (FF = 0.28) (Table S1, ESIz).
This can be attributed to the presence of ferrocenyl moieties, which
effectively quench the singlet excited state of the phthalocyanines
by photoinduced electron transfer.8
Fig. 2 Comparison of the rate of photodegradation of DPBF (initial
concentration = 70 mM) sensitised by 6a (4 mM) upon exposure
to different concentrations of DTT for 24 h in PBS with 0.5%
Cremophor EL. The dark control is shown as closed symbols.
The singlet oxygen quantum yields (FD) of these phthalo-
cyanines in DMF were also determined by a steady-state method
using 1,3-diphenylisobenzofuran (DPBF) as the scavenger. Fig. S2
(ESIz) compares the rate of photodegradation of DPBF sensitised
by these compounds and the unsubstituted zinc(II) phthalocyanine
(ZnPc) used as a reference. The values of FD are also listed in
Table S1 (ESIz). Similarly, the values of 6a (FD = 0.17) and 6b
(FD = 0.13) are significantly lower than that of 7 (FD = 0.34),
which again can be explained by the reductive quenching by the
ferrocenyl moieties, which disfavours the intersystem crossing and
eventually the formation of singlet oxygen.
analogue 6b, the fluorescence intensity was only increased slightly
even in the presence of 40 mM of DTT (Fig. S3b, ESIz and Fig. 1).
The absorption spectra of 6a and 6b were also recorded
upon exposure to different concentrations of DTT over 24 h
(Fig. S4, ESIz). The sharp Q band was virtually unchanged in
all the cases, which suggested that both compounds and the
phthalocyanine fragment of 6a remained non-aggregated, and
the fluorescence enhancement for 6a was due to relaxation of the
quenching effect upon reductive cleavage of the disulfide linker
rather than the change in aggregation state.
To demonstrate that 6a is responsive towards reducing stimuli,
we recorded the fluorescence spectra of 6a in phosphate buffered
saline (PBS) in the presence of 0.5% Cremophor EL and different
concentrations of DTT (0–40 mM) over 24 h (Fig. S3a, ESIz). As
shown in Fig. 1, the fluorescence intensity increases with time and
the concentration of DTT. In the absence or presence of 2 mM of
DTT, which mimics the extracellular reducing environment, the
fluorescence intensity only increases slightly. However, upon
addition of 10 or 40 mM of DTT, which mimics the intracellular
reducing environment, particularly in tumour, the fluorescence
intensity increases substantially within the first 6 h. This can be
attributed to the cleavage of the disulfide linker by DTT preventing
quenching by the ferrocenyl moieties. For the non-cleavable
DTT-mediated production of singlet oxygen by these com-
pounds was also investigated. Fig. 2 compares the rate of
photodegradation of DPBF sensitised by 6a upon exposure to
different concentrations of DTT for 24 h in PBS. In the dark, the
compound could not produce singlet oxygen even in the presence
of 10 mM of DTT. Upon illumination and at up to 2 mM of DTT,
the rate of singlet oxygen production was still very slow. However,
when the concentration of DTT was increased to 5 or 10 mM, the
singlet oxygen production efficiency was remarkably enhanced.
Clearly, the photosensitisation ability of 6a was restored upon
reductive cleavage of the disulfide linker. This was supported by the
response of 6b, for which the rate of singlet oxygen production
remained slow even in the presence of 10 mM of DTT. By contrast,
for the non-ferrocene-containing reference compound 7, the
efficiency was remarkably high and very similar to that of 6a under
the same conditions (Fig. S5, ESIz).
The therapeutic efficacy of all these compounds was investigated
against MCF-7 human breast cancer cells. Fig. S6 (ESIz) shows the
dose-dependent survival curves for phthalocyanines 6a, 6b and 7,
and the ferrocenyl chalcone analogue 5a after incubation for 6 or
24 h and with prior treatment with different concentrations of DTT
for 1 h, both in the absence and presence of light. All these
compounds were non-cytotoxic in the dark. Upon illumination
(l > 610 nm, 40 mW cmꢀ2, 48 J cmꢀ2) and without prior
treatment with DTT, 6a and 6b exhibited similar photocytotoxicity.
Both of them were less potent than 7 probably due to the
quenching effect by the ferrocenyl units. Unexpectedly, compound
5a was not cytotoxic up to a concentration of 1 mM. Similar results
were obtained after the cells were pre-treated with 2 mM of DTT.
However, when 4 mM of DTT was used, the photocytotoxicity of
Fig. 1 Changes in fluorescence intensity of 6a (closed symbols) and
6b (open symbols) upon exposure to 0 mM (circles), 2 mM (squares),
10 mM (triangles) and 40 mM (stars) of DTT in PBS with 0.5%
Cremophor EL.
c
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Table 1 Comparison of the IC50 values of 6a, 6b and 7 against
MCF-7 cells after incubation for 6 or 24 h and with prior treatment
with different concentrations of DTT for 1 h
To reveal the subcellular localisation of 6a after disulfide
cleavage, we coincubated the MCF-7 cells, which were
pre-treated with DTT (4 mM) for 1 h, with 6a and ER-Tracker
Green, LysoTracker Green DND 26 or MitoTracker Green
FM, which are specific fluorescent dyes for endoplasmic
reticulum, lysosomes and mitochondria respectively. As
shown in Fig. S8 (ESIz), the fluorescence caused by the
ER-Tracker can superimpose with the fluorescence caused
by 6a after disulfide cleavage. This observation suggests
that the phthalocyanine fragment of 6a is localised in the
endoplasmic reticulum of the cells. By contrast, the fluorescence
images could not be overlapped for the cases of MitoTracker
and LysoTracker (Fig. S9, ESIz), indicating that the fragment is
not preferentially localised in the mitochondria and lysosomes
of the cells.
IC50 (nM)
6 h Drug incubation
24 h Drug incubation
0 mM
DTT
2 mM
DTT
4 mM
DTT
0 mM
DTT
2 mM
DTT
4 mM
DTT
6a 160 ꢁ 5 150 ꢁ 5
81 ꢁ 5 124 ꢁ 2 119 ꢁ 2
50 ꢁ 1
6b 179 ꢁ 8 162 ꢁ 4 163 ꢁ 6 149 ꢁ 6 135 ꢁ 5 130 ꢁ 9
7
80 ꢁ 7
75 ꢁ 6
76 ꢁ 6
52 ꢁ 2
51 ꢁ 2
50 ꢁ 2
6a was significantly enhanced, while that of 6b and 7 remained
essentially unchanged. Table 1 compares their IC50 values, defined
as the drug concentration required to kill 50% of the cells, under
different conditions. It can be seen that only 6a is DTT-responsive.
For 24 h drug incubation, pre-treatment with 4 mM of DTT
reduced its IC50 value by more than 2-fold from 124 to 50 nM,
which was almost the same as that of 7. On the basis that the
intracellular thiol level is in millimolar range and tumour tissues
generally have an elevated thiol concentration compared with
normal tissues,13 these results demonstrate that 6a is a promising
photosensitiser for targeted PDT, which can be activated in a
reducing environment, such as tumour, leading to enhanced
fluorescence emission, singlet oxygen production and in vitro
photodynamic activity.
In summary, we have prepared a novel disulfide-linked
ferrocenyl phthalocyanine which can be activated by DTT at
millimolar concentrations. On the basis that the intracellular
thiol level is in this range and tumour tissues generally have an
elevated thiol concentration compared with normal tissues, this
compound is a promising photosensitiser for targeted PDT.
We thank Prof. Wing-Ping Fong and Sin-Lui Yeung for
performing the confocal microscopic study. This work was
supported by a grant from the Research Grant Council of the
Hong Kong Special Administrative Region (project no.
402211).
To further investigate the reductive disulfide cleavage of 6a
at the cellular level, the intracellular fluorescence of this
compound was examined under different conditions. MCF-7
cells were first treated with different concentrations of DTT for
1 h followed by incubation with 6a for 24 h. As shown in
Fig. 3a, the cells being pre-treated with 4 mM of DTT show
strong intracellular fluorescence, while the fluorescence is
hardly observed for the cases of 0 and 2 mM of DTT. For
the non-cleavable analogue 6b, the intracellular fluorescence
remained extremely weak for all the three cases (Fig. S7, ESIz).
The average relative fluorescence intensity per cell for these
compounds was also measured and is compared in Fig. 3b.
Only 6a at high concentration of DTT (4 mM) shows high
intracellular fluorescence, which is about 9-fold higher than
that of the other cases. These results are also in accord with the
cleavage of the disulfide linkage in 6a by DTT inside the cells.
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Fig. 3 (a) Confocal fluorescence images of MCF-7 cells pre-treated
with different concentrations of DTT followed by incubation with 6a
(1 mM) for 24 h (lower row). The corresponding bright field images are
shown in the upper row. (b) Comparison of the intracellular fluorescence
intensity for 6a and 6b at different concentrations of DTT. Data are
expressed as mean ꢁ standard deviation (number of cells = 30).
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun.