A novel strategy for targeting photodynamic therapy. Molecular combo of photodynamic agent zinc(II) phthalocyanine and small molecule target-based anticancer drug erlotinib

Article abstract of DOI:10.1039/c3cc45487h

In this study, two phthalocyanine-erlotinib conjugates linked by an oligoethylene glycol chain have been synthesised and fully characterised. Having erlotinib as the targeting moiety, the two conjugates exhibited high specific affinity to HepG2 cancer cells and tumour tissues, therefore leading to high photodynamic activity. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc45487h

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A novel strategy for targeting photodynamic therapy.
Molecular combo of photodynamic agent zinc(II)
phthalocyanine and small molecule target-based
anticancer drug erlotinib†
Cite this: Chem. Commun., 2013,
49, 9570
Received 19th July 2013,
Accepted 16th August 2013
DOI: 10.1039/c3cc45487h
Feng-Ling Zhang,z Qi Huang,z Ke Zheng, Jun Li, Jian-Yong Liu* and Jin-Ping Xue*
www.rsc.org/chemcomm
In this study, two phthalocyanine–erlotinib conjugates linked by makes its accuracy, a basic demand for drug administration,
an oligoethylene glycol chain have been synthesised and fully much lower than PSs possessing targeting properties inherently.10
characterised. Having erlotinib as the targeting moiety, the two For the latter, the complicated structure and low stability of
conjugates exhibited high specific affinity to HepG2 cancer cells and biomacromolecules always induce a difficult synthesis and pur-
tumour tissues, therefore leading to high photodynamic activity.
ification process, more importantly, their biological activities are
often changed, even lost during the process of modification.11
Small molecule target-based cancer therapy that aims to specific
Photodynamic therapy (PDT) is an increasingly valuable thera-
peutic modality for a range of tumours and non-malignant affinity to cancer cells by modulating the aberrant molecular
diseases. In the PDT process, the combined action of a photo- pathways underlying tumour growth and progression has achieved
sensitizer (PS), appropriate light and molecular oxygen is used to tremendous success in recent decades.12 Gefitinib and erlotinib
generate cytotoxic reactive oxygen species (ROS) and cause can target the ATP binding domain of tyrosine kinase in EGFR
destruction of tumours.1 Compared with conventional therapies, (epidermal growth factor receptor); overexpressed tumours have
PDT holds the promise of dual selectivity, which could be fulfilled shown specific affinity to tumour cells, thereby leading to targeting
by restricting the illumination to a specific region and preferential cancer therapy.13 It is envisioned that PSs covalently binding with
tumour uptake of the PS.2 To date, most of the first- and second- such small molecule target-based anticancer drugs may combine
generation PSs studied for PDT have exhibited only a slight the high therapeutic efficiency of PSs and excellent specificity of
preference for malignant cells, which brings about significant small molecule target-based anticancer drugs.
skin photosensitivity and high uptake by healthy cells and
In this communication, we choose erlotinib as the target
tissues.2 In recent years, much effort has been devoted to the moiety to conjugate with a zinc(II) phthalocyanine core through
development of third-generation PSs for targeting photodynamic an oligoethylene glycol spacer which can improve the amphi-
therapy.3 There are several approaches to establish this kind of PS. philicity and biocompatibility of the conjugate. The developed
One is making use of nanoparticles modified with target moieties phthalocyanine–erlotinib conjugates display targeting photo-
as delivery vehicles for PSs.4 Another is conjugation of second- dynamic activities against HepG2 cancer cells and specific
generation PSs to biomacromolecules with targeting function affinity to tumour tissues in nude mice. To the best of our
such as antibody,5 lipoprotein,6 peptide,7 transferrin,8 aptamer9 knowledge, there are few reports on the small molecule target-
etc. Promising results and decades of progress have been achieved based anticancer drugs conjugated with PDT agents.
by these two ways, but they also faced some challenges. For the
Synthesis of the phthalocyanine–erlotinib conjugates 3a–3b
former, the complex control procedure over the size, shape, is shown in Scheme 1. Firstly, azide (oligoethylene glycol) under-
stability, drug loading and releasing capacity of nanoparticles went 1,3-dipolar cycloaddition with erlotinib in the presence of
sodium ascorbate and CuSO4ꢀ5H2O to afford 1a–1b (54–63%).
Treatment of these two compounds with 3-nitrophthalonitrile
gave phthalonitriles 2a–2b in 64–72% yield, which then under-
College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou,
350108, P. R. China. E-mail: liujianyong82@163.com, xuejinping66@fzu.edu.cn;
went statistical condensation with unsubstituted phthalonitrile
Fax: +86-591-87892632; Tel: +86-591-83758209
† Electronic supplementary information (ESI) available: Experimental details, using DBU (1,8-diazabicyclo-[5.4.0]undec-7-ene) as a base to afford
synthesis of phthalocyanine 4, UV-Vis spectra of 3a–4 in DMF and cell culture
‘‘3+1’’ products 3a–3b (23–25%). For comparison, phthalocyanine 4
medium, cytotoxic effects of 3a–4 toward HepG2 cells, in vitro ROS generation for
without the erlotinib moiety was synthesised as a reference com-
3a–4, confocal fluorescence images of HepG cells after incubation with 3a–4, and
1H, 13C{1H} NMR, and HRMS spectra of all the new compounds. See DOI: 10.1039/
pound according to the previous procedure (Scheme S1, ESI†).14
Because of the large-conjugated structure, phthalocyanine
derivatives always tend to aggregate. The aggregated phthalocyanines
c3cc45487h
‡ F.-L. Zhang and Q. Huang contributed equally to this work.
c
9570 Chem. Commun., 2013, 49, 9570--9572
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at 670 nm, they exhibited considerable cytotoxicity. The IC50
values of 3a and 3b were determined to be 0.01 and 0.04 mM,
respectively, with the light dose of 1.5 J cmꢁ2. The values are
roughly equivalent to that of phthalocyanine 4. The high
photodynamic activities agree well with their very low aggrega-
tion in the culture medium. The phototoxicity of erlotinib was
also investigated under the same conditions, but no photo-
cytotoxicity was observed up to 0.5 mM.
The excitation of the photosensitizer results in the genera-
tion of ROS, which is thought to be the main mediator of
cellular death induced by PDT. It can mediate cellular effects
such as lipid peroxidation and vascular effects, leading to direct
or indirect cytotoxic effects on the treated cells. Generally, the
higher the ROS, the higher the photocytotoxicity. Here, the ROS
generation efficiency of all the phthalocyanines against HepG2
cells was also investigated (Fig. S4, ESI†). It follows the order
3a > 4 > 3b which is in accordance with their FD in DMF and
in vitro phototoxicity. The results reveal that the oligoethylene
glycol chain and the erlotinib moiety may have an effect on the
ROS generation efficiency and photodynamic activity of phthalo-
cyanines to a certain extent.
The subcellular localisation of 3a, 3b and 4 was studied
using an Olympus FV 1000 Confocal laser scanning micro-
scope. The cells were first incubated with the phthalocyanines
in the culture medium for 24 h, and then stained with Lyso-
Tracker DND 26 (for 60 min) or MitoTracker Green FM (for
30 min), which are specific dyes for lysosomes and mitochon-
dria, respectively. As shown in Fig. S5a (ESI†), the fluorescence
caused by the LysoTracker (excited at 488 nm, monitored at
510–570 nm) or the MitoTracker (excited at 488 nm, monitored
at 510–570 nm) can superimpose with the fluorescence caused
by 3a (excited at 633 nm, monitored at 650–750 nm). The very
similar fluorescence intensity line profiles (Fig. S5b, ESI†) of 3a
and LysoTracker or MitoTracker traced along the white lines in
Fig. S5a (ESI†) also indicate that 3a can be localised in the
lysosomes and mitochondria of the cells. The very similar
subcellular localisations of 3b and compound 4 were observed
(Fig. S6 and S7, ESI†). The results showed that these three PSs
had no organelle specificity and were distributed throughout
the cytoplasm. This may induce the all-around destruction of
cancer cells after PDT treatment with these PSs.
Scheme 1 Synthesis of phthalocyanine–erlotinib conjugates.
exhibit no photocytotoxicity.15 The absorption spectra of phthalo-
cyanines 3a and 3b showed a strong and sharp Q-band at 678 nm
suggesting that they were dissolved well and almost did not
aggregate in DMF (Fig. S1, ESI†). To account for the in vitro
photodynamic activities of compounds 3a and 3b, their absorp-
tion spectra in the culture medium (RPMI 1640) were also
recorded (Fig. S2, ESI†), which are very similar to those of 3a
and 3b in DMF. Their singlet oxygen quantum yields (FD) and
fluorescence quantum yields (FF) in DMF were also determined
and are listed in Table 1. Both 3a and 3b display comparative FD
and FF to the reference compound 4. This indicates that the
photophysical properties of the phthalocyanines did not change
obviously after conjugation with erlotinib.
To evaluate the photosensitizing efficiency of these conju-
gates, the HepG2 cells which overexpress EGFR were selected
and a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) cell viability assay was employed. The cytotoxi-
city of these compounds toward HepG2 cells is shown in Fig. S3
(ESI†) and the IC50 values are summarized in Table 2. It can be
seen that all these phthalocyanines do not show obvious dark
cytotoxicity up to 50 mM. However, upon illumination with light
Table 1 Photophysical/photochemical data of phthalocyanines in DMF
b
c
Compounds
lmax/nm (log e)
lema/nm
FF
FD
To assess the specificity of the conjugates to the cancer cells,
we mixed the HELF cells (human embryo lung fibroblasts, a cell
line with low expression of EGFR) and HepG2 cells which have
huge morphological differences from each other in a cell culture
dish. After incubation with phthalocyanines for 24 h, the fluores-
cence caused by 3a, 3b or 4 (all excited at 633 nm, monitored at
650–750 nm) in these two cell lines was recorded using a confocal
laser scanning microscope (Fig. 1a). The fluorescence of 3a and 3b
in HepG2 cancer cells is obviously brighter than in HELF
(approximately 3 fold). However, there is no obvious difference
between HELF and HepG2 cells for reference 4 (Fig. 1b). The
results indicate that 3a and 3b can successfully target the cancer
cells with high expression of EGFR. The fluorescence ratio of 3a
among the two cell lines is equivalent to that of 3b indicating that
the oligoethylene glycol chain length did not influence the target
ability of erlotinib.
3a
3b
4
678 (5.25)
678 (5.28)
677 (5.41)
685
686
684
0.27
0.27
0.26
0.66
0.57
0.63
a
b
Excited at 610 nm. Using unsubstituted zinc(II) phthalocyanine
c
(ZnPc) in DMF as the reference (FF = 0.28). Using ZnPc in DMF as
the reference (FD = 0.56).
Table 2 IC50 values for phthalocyanines 3a, 3b and 4 against HepG2 cancer cells
with the light dose of 1.5 J cmꢁ2
Compounds
IC50 (mM)
3a
3b
4
0.01
0.04
0.03
N a
Erlotinib
a
Noncytotoxic up to 0.5 mM.
c
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toward HepG2 cancer cells and A431 tumour tissues. The
conjugates contain both photodynamic and targeting anti-
cancer therapy agents which are covalently linked and function
in a cooperative manner. The introduction of the erlotinib
moiety can enhance the specificity of phthalocyanine units to
HepG2 cancer cells and A431 tumour tissues. The IC50 value of
the conjugates is as low as 0.01 mM toward the HepG2 cells,
which is equivalent to that of the reference compound 4 without
the erlotinib derivative. The tumour/skin ratio of the conjugate
is actually about 5-fold higher than that of reference 4. The
overall results show that the conjugates are highly promising
antitumour agents for dual targeting and photodynamic therapy.
This may provide a novel targeting strategy for PDT, as well as
other cancer therapy modalities.
Fig. 1 Confocal fluorescence images of mixed HepG2 and HELF cells after
incubation with 3a, 3b and 4 for 24 h (all at 10 mM); (b) comparison of relative
intracellular average fluorescence intensity of phthalocyanines in HepG2 and
HELF cells (measured in the ROIs). Data are expressed as means ꢂ SD. Statistical
significance **(P o 0.01).
We thank the Natural Science Foundation of China (Project
No. 21101028), the Major Project of the State Ministry of
Science and Technology of China (Project No. 2011ZX09101-
001-04), and the Natural Science Foundation of Fujian Province
(Project No. 2012J05021) for financial support.
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