In vitro and in vivo evaluation of improved EGFR targeting peptide-conjugated phthalocyanine photosensitizers for tumor photodynamic therapy

Article abstract of DOI:10.1016/j.cclet.2018.04.025

Phthalocyanine (Pc) is one of the most promising photosensitizer candidates because of its strong absorption (extinction coefficient ε > 10⁵ L mol^?1 cm^?1) at long wavelengths and strong singlet oxygen generation abilities

Full text of DOI:10.1016/j.cclet.2018.04.025

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Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
In vitro and in vivo evaluation of improved EGFR targeting
peptide-conjugated phthalocyanine photosensitizers for tumor
photodynamic therapy
Qingle Chena,b,1, Yanhong Mab,1, Jisi Zhaob, Mei Zhaob, Wenjing Lib, Qian Liub,
a,
b,
Li Xiongc, , Wenjie Wu , Zhangyong Hong
*
*
*
a
College of Materials Science and Chemical Engineering, Tianjin University of Science and Technology, Tianjin 300457, China
State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Sciences, College of Life Sciences, Nankai University, Tianjin 300071,
b
China
c
Department of General Surgery, Second Xiangya Hospital, Central South University, Changsha 410011, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Phthalocyanine (Pc) is one of the most promising photosensitizer candidates because of its strong
Available online xxx
absorption (extinction coefficient e
> 105 L molꢀ1 cmꢀ1) at long wavelengths and strong singlet oxygen
generation abilities (a singlet oxygen quantum yield of approximately 50%). However, low tumor
targeting, low water solubility and a high tendency to aggregate appear to significantly restrict the
compound’s application in tumor treatment. Conjugating Pc with peptide ligands could be a useful
strategy for alleviating these problems. Here, to further optimize the structures of peptide-conjugated
zinc Pcs for PDT therapy, we finely tuned the hydrophilicity of the modified Pc aromatic macrocycle with
varied length of polyethylene glycol (PEG) and added an extra PEG linker and an extra glutamic acid
between the Pc ring and the peptide ligand to reduce the influence of the ligand on the Pc aromatic ring.
Among the synthesized conjugates, Pc-3 showed greatly improved targeting towards tumors and
abolished inoculated tumors with only a single PDT treatment in a subcutaneous xenograft tumor model,
making this approach a promising therapeutic agent for the treatment of cancer.
Keywords:
Photodynamic therapy
Potosensitizer
Phthalocyanine
Peptide conjugate
© 2018 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Photodynamic therapy (PDT) has emerged as an important non-
invasive therapeutic modality in the management of a variety of
premalignant and malignant diseases [1]. However, the limited
availability of ideal photosensitizers with desired properties
continues to be an important bottleneck in PDT therapy [2]. In
recent decades, substantial effort has been made toward the
development of new photosensitizers for PDT [3]. Among them,
phthalocyanine (Pc) is one of the most promising candidates [4]. Pc
has advantageous photophysical and photochemical properties,
hydrophobic nature of the planar Pc macrocycle structure [6],
which always renders Pcs photodynamically inactive in aqueous
media [7] and significantly restricts their in vivo biological and
medical applications. Many studies have reported on the chemical
modification of Pcs through the attachment of hydrophilic
substituents to peripheral positions on the macrocycle to increase
the water solubility of Pcs [8,9]. However, hydrophilic modification
greatly reduces the PDT activity of Pcs, possibly by reducing their
cell permeability and membrane attachment [3b].
Conjugating Pcs to peptide ligands could be another practical
and useful strategy for improving the physical properties of Pcs
[10,11]. Conjugation with proper hydrophilic tumor-homing
peptide ligands can increase water solubility and reduce the
aggregation of Pcs. It is also possible to improve Pcs’ ability to
target tumors through interactions between receptors and the
peptide ligands [12]. However, most studies have used non-
modified or alkyl-group-modified Pcs with which to conjugate
peptide ligands. In this context, the problems associated with the
strong hydrophobicity of Pcs, such as aggregation and low water
solubility, cannot be well resolved by peptide conjugation. In
including strong absorption (extinction coefficient
e
> 105 L molꢀ1
cmꢀ1) at long wavelengths (>670 nm) and strong singlet oxygen
generation abilities (a singlet oxygen quantum yield of approxi-
mately 50%) [5]. However, most Pcs exhibit extremely low
solubility and strong tendency to aggregate in water due to the
* Corresponding authors.
E-mail addresses: lixionghn@163.com (L. Xiong), wwjie@tust.edu.cn (W. Wu),
hongzy@nankai.edu.cn (Z. Hong).
1
These two authors contributed equally to this work.
https://doi.org/10.1016/j.cclet.2018.04.025
1001-8417/© 2018 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Q. Chen, et al., In vitro and in vivo evaluation of improved EGFR targeting peptide-conjugated phthalocyanine
photosensitizers for tumor photodynamic therapy, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.04.025

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addition, the strong hydrophobicity of Pcs enhances non-specific
interactions with the cell membrane and destroys the binding
function of the peptide ligands, increasing the non-specific
background signal.
varying the hydrophilic substitutions on the Pc ring and by adding
a hydrophilic linker to finely tune the hydrophilicity, with the aim
of balancing the tumor selectivity and PDT activity of the
conjugates.
In our previous studies [13], using hydrophilic group-modified
Pcs (i.e., modification with triethyleneglycol monomethyl ether
substitution or glycol chain substitution) to construct peptide-Pc
conjugates, the conjugates were well-shielded from non-specific
binding with cells and showed improved tumor selectivity in cell-
based experiments. However, the PDT efficiency obtained in the
cell-based experiments was appreciably reduced after making
these modifications, particularly for the relatively highly hydro-
philic modification (glycol chain substitution). Compared with the
relatively less hydrophilic modification, the highly hydrophilic
modification appears to increase tumor selectivity and reduce the
background distribution in vivo but with relatively low PDT
efficiency. This disparity may require us to finely tune the
hydrophilicity of peptide-conjugated Pcs to obtain a better PDT
outcome. Moreover, the PDT potential of such Pcs for in vivo tumor
treatment has not been tested in animal models. Thus, we set out to
further optimize the structure of peptide-conjugated Pcs by
We designed a solid-phase strategy [14] for the synthesis of the
proposed conjugates. For comparison, four hydrophilic substitu-
tions (ethylene glycol monomethyl ether group, diethylene glycol
monomethyl ether group, triethylene glycol monomethyl ether
group and hexaethylene glycol monomethyl ether group) were
adopted to reduce the hydrophobicity of the Pc ring component
(Pc-1-4 in Fig. 1). Asymmetrically substituted A3B-type Pcs with
hydrophilic substitutions on the peripheral positions of the
macrocycle and a single carboxylic acid were designed to facilitate
convenient conjugation with amine-containing peptide ligands. An
extra polyethylene glycol (PEG) chain [15] and an extra glutamic
residue (Pc-5 in Fig. 1) were incorporated between the Pc
macrocycle component and peptide ligand to test their effect in
further increasing water solubility of the conjugates and reducing
the influence of the Pc aromatic macrocycle on the affinity of the
peptide ligands. The short peptide Lys-Ala-Arg-Leu-Leu-Thr, a
modified EGF receptor-targeting peptide [13b,16], was coupled to
Fig. 1. Design of EGFR targeting peptide-conjugated Pcs. Pc-1-4 feature ethylene glycol chains of varying lengths on the PC ring component. Pc-5 features an extra aspartic
acid residue on the linker compared with Pc-3.
Please cite this article in press as: Q. Chen, et al., In vitro and in vivo evaluation of improved EGFR targeting peptide-conjugated phthalocyanine
photosensitizers for tumor photodynamic therapy, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.04.025

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Fig. 2. Structure of Pc-Control, with a disorderly peptide sequence and no EGFR targeting capability compared with Pc-3.
Pcs for tumor-targeting purposes. Pc-Control (in Fig. 2), which
contained the LLKATR ligand (a disorderly sequence of the EGFR
targeting ligand KARLLT with no affinity to EGFR receptor), was
synthesized as a control.
carboxyl group of Pc 11, 12, 13 or 14 using 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-
benzotriazole (HOBT) as the activating agents. The peptide-Pc
conjugate was cleaved from the resin by removing all protecting
groups at the same time in 95% TFA with triisopropylsilane (TIS)
(TFA:H2O:TIS = 95:2.5:2.5) to obtain the desired conjugates Pc-1,
Pc-2, Pc-3 and Pc-4 after HPLC purification. Similarly, Pc-5 and Pc-
Control were prepared by coupling Pc 13 with side chain-
The synthetic route to the above mentioned Pc conjugates is
shown in Scheme 1. Phthalonitrile 6 was submitted to statistical
condensation with phthalonitrile 7, 8, 9 or 10 in n-hexanol at 160 ꢁC
for 7 h in the presence of Zn(II) acetate and 1,8-diazabicyclo[5.4.0]
undec-7-ene (DBU), followed by hydrolysis with NaOH in THF/
water/methanol (2/1/5) and subsequent acidification. The reaction
afforded Pc 11, 12, 13 and 14 with a free carboxylic group for
peptide conjugation. The yields for these Pcs typically ranged from
15% to 30% in two steps. The peptide NH2-Linker-Lys(Boc)-Ala-Arg
(Pbf)-Leu-Leu-Thr(tBu)-resin 15 with protected side chains was
prepared on a resin using a Fmoc solid-phase peptide synthesis
(SPPS) protocol. The N terminus of peptide 15 was coupled to the
protected
NH2-Glu(tBu)-Linker-Lys(Boc)-Ala-Arg(Pbf)-Leu-Leu-
Thr(tBu)-resin (16) or sequence-disordered NH2-Glu(tBu)-Linker-
Arg(Pbf)-Thr(tBu)-Ala-Lys(Boc)-Leu-Leu-resin (17) via solid-phase
synthesis, followed by deprotection in 95% TFA (TFA:H2O:TIS =
95:2.5:2.5) in good yield after HPLC purification.
The UV–vis absorption spectra (Fig. 3 and Table 1) of the
synthesized Pc conjugates were obtained in solvents, DMSO, PBS, or
PBS containinga lowconcentration of lipids. In DMSO, the electronic
Scheme 1. Synthesis of the designed peptide conjugated Pcs.
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photosensitizers for tumor photodynamic therapy, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.04.025

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Fig. 3. UV–vis absorption spectra of Pc-1 (A), Pc-2 (B), Pc-3 (C), Pc-4 (D) and Pc-5 (E) in various solvents (i.e., DMSO, 0.05% Tween 80 in PBS and PBS solution). The
concentration for absorption determination was 10 mmol/L.
Table 1
Table 2
Absorption spectra data of Pc-1-5 in DMSO, 0.05% Tween 80 in PBS and PBS. Data are
expressed as lmax/nm (log ).
Photophysical and photochemical parameters of Pc-1-5 in DMSO.
e
Compound
FD
lex (nm)
lem (nm)
Dstokes (nm)
Solution
lmax/nm (log
Pc-1 Pc-2
681 (5.01) 681 (4.96) 680 (4.97) 680 (4.90) 681 (5.03)
e)
Pc-1
Pc-2
Pc-3
Pc-4
Pc-5
0.56
0.60
0.54
0.49
0.56
686
684
683
683
683
689
688
689
688
689
3
4
6
5
6
Pc-3
Pc-4
Pc-5
DMSO
PBS + Tween 80 680 (4.66) 680 (4.55) 680 (4.64) 680 (4.84) 681 (4.84)
PBS 677 (4.07) 678 (4.09) 679 (4.04) 679 (4.09) 678 (4.05)
FD: singlet oxygen quantum yield. For ZnPc, FD = 0.67.
spectra of all five synthesized Pc conjugates showed intense
absorption in the Q band region at 680–681 nm, which is redshifted
by approximately 10 nm compared with the Q band region of non-
substituted PcZn (ꢂ670 nm). The conjugates also showed almost
completely monomeric behavior, as indicated by a single, narrow Q
band. All conjugates exhibited excellent solubility in aqueous
solution, while they formed cofacial aggregates in PBS, as evidenced
by the higher-energy (blue shifted) band at 620–640 nm in the Q
band region. With the addition of 0.05% Tween 80 to the aqueous
695 nm, which are typical for metal Pc complexes. The singlet
oxygen quantum yields of these conjugates were determined in
DMSO, as shown in Table 2. All five Pc conjugates showed roughly
the same singlet oxygen quantum yield, in the range 0.49–0.60,
comparable to that of non-substituted ZnPc (approximately 0.67).
This finding indicates that peptide conjugation and Pc ring
modification have only a very moderate effect on singlet oxygen
quantum yield. Singlet oxygen (1O2) is believed to be the major
cytotoxic agent involved in PDT, and a high singlet oxygen quantum
yield should be highly important for photodynamic therapy
(Table 3).
solution, the Q band at
l= 600–700 nm became much sharper, and
the spectral shape became similar to that in DMSO. This finding
indicated that the addition of a very low concentration of lipids can
effectively break up the aggregates in aqueous solution. The
aggregation of Pc-4 with hexaglycol substitution and that of Pc-5
with an extra glutamic acid residue were relatively more easily
disrupted, as demonstrated by the relatively sharper and stronger
peak observed at 680 nm. The hydrophilic ethylene glycol chain
surrounding the Pc rings, hydrophilic peptide ligand and hydro-
philic PEG linker significantly contributed to relieving the
aggregation problem associated with zinc Pcs.
Table 3
Phototoxicity of Pc conjugates determined by MTT assay.
Compound
IC50 (nmol/L)
A431 cells
A549 cells
MCF-7 cells
PC-3 cells
Pc-1
Pc-2
Pc-3
Pc-4
Pc-5
Pc-Control
380
240
220
190
960
1950
220
740
170
320
850
2090
240
130
520
230
970
2400
280
330
650
440
1290
2290
Fluorescence spectra of the Pc conjugates were obtained in
DMSO (Table 2 and Fig. S3 in Supporting information). All
synthesized Pcs displayed a fluorescence excitation band at
approximately 685 nm and an emission band at approximately
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The in vitro PDT activity of Pc-1-5 was evaluated by measuring
cellular survival rates in human tumor cell lines, including A431
and A549 cells, which express very high levels of EGFR, and MCF-7
and PC-3 cells, but exhibit relatively low expression levels of EGFR,
via the MTT assay (Fig. 4). The results show that all five Pc
conjugates exhibited substantial cytotoxicity when treated by
irradiation for 10 min with a 660-nm laser. Among the conjugates,
Pc-3 displayed the highest PDT activity and selectivity. Lower EC50
(50% inhibition of cell proliferation based on dose-response
curves) levels were observed for A431 and A549 cells, approxi-
mately 220 nmol/L and 170 nmol/L, respectively, than for MC-7 and
PC-3 cells, approximately 520 nmol/L and 650 nmol/L, respectively.
Interestingly, Pc-5 showed an EC50 value of approximately 1000-
1200 nmol/L towards these tumor cells, much higher than those of
Pc-3. An extra carboxylic acid group in the linker appears to
significantly reduce the PDT activity of the Pc conjugates. The
negative charge of the carboxylic group may reduce the association
of the conjugate towards the plasma or organellar membrane, thus
reducing PDT activity. When the cells were treated with the
conjugate Pc-Control—prepared by conjugation of Pc 13 with a
disorderly sequence of EGFR peptide and no affinity toward EGFR
receptor—we found much lower PDT activity towards these tumor
cells, with EC50 values of 1900, 2100, 2400 and 2200 nmol/L for
A431, A549, MCF-7 and PC-3 cells, respectively (Fig. 5A). The PDT
activities towards A431 and A549 cells expressing high levels of
EGFR receptor were reduced approximately ten-fold compared
with the PDT activity of Pc-3 (1900 nmol/L, 2100 nmol/L /
220 nmol/L, 170 nmol/L, respectively). These results indicate that
peptide targeting is highly important for the PDT activity of these
Pc conjugates. All conjugates were essentially non-cytotoxic in the
dark toward all cell lines (Fig. 5B), and a cell viability of 90% was
observed without irradiation after incubation with these con-
jugates at concentrations of up to 3000 nmol/L, indicating that
these conjugates show very good biocompatibilities without
exposure to irradiation.
Fig. 5. (A) Phototoxicity efficacy of Pc-Control, with disorderly peptide sequence,
toward A549, A431, MCF-7 and PC-3 cells after cells were treated with a series of
concentrations of the compound and illuminated for 10 min (laser wavelength of
660 nm and laser power density of 40 mW/cm2). (B) Dark toxicity of Pc-1, Pc-2, Pc-
3, Pc-4 and Pc-5 toward A431 cells without light illumination.
while those in the normal tissues began to decrease rapidly. All Pcs
showed good tumor accumulation, and enrichment consistently
occurred within several hours of injection. Compared with other
three Pcs, Pc-3 and Pc-5 showed relatively better tumor-targeting
capabilities. The mice injected with Pc-3 or Pc-5 showed quick
accumulation of the intense fluorescence signal in the tumor area
and a rapid decrease in the background fluorescence signal in
normal tissue. At only 2 h post-injection, the signals were located
almost entirely in the tumor area; negligible signals were detected
in other tissues, which resulted in excellent fluorescence imaging
contrast that could clearly distinguish tumors from the surround-
ing normal tissue. The intense fluorescence signal in the tumor site
could be maintained for more than 12 h post-injection. The mice
injected with Pc-1 and Pc-2 also showed a very strong fluorescence
signal in the tumor site but relatively stronger background
fluorescence signals in normal tissues, particularly in the liver.
This lower contrast may be due to the relatively higher
hydrophobicity of the Pc component. The mice injected with Pc-
4 showed much slower clearance of the background fluorescence
signals than did the other mice. The relatively long hexaethylene
glycol monomethyl ether substitution near the Pc component may
afford the conjugate a longer plasma half-life, and longer times
were required for the non-enriched conjugate to be filtered from
the plasma. The in vivo distribution results may indicate that Pc-3
and Pc-5 have relatively better enrichment capabilities. Both of
them can well accumulate in tumor sites with weak background
intensity, and may have good potential for in vivo tumor imaging
and tumor-targeted PDT.
To verify the phototoxicity of these conjugates visually, the
morphological changes in A431 cells after PDT treatment with
these conjugates were observed using microscopy. As shown in
Fig. 6, without PDT treatment, the cells showed unabridged shapes,
while the cells treated by incubation with conjugates, followed by
irradiation for 10 min with a 660 nm-laser, showed significant
morphological changes, including cellular swelling, bleb forma-
tion, and vesicle rupture. The PDT activities of these conjugates
were distinct.
The in vivo distributions of the conjugates in tumor-bearing
mice were monitored continuously with an in vivo fluorescence
imaging system (IVIS Lumina II, Xenogen, Alameda, CA, USA). The
strong NIR fluorescence of the Pc component greatly facilitated the
monitoring. As shown in Fig. 7, all Pcs injected into the mice were
quickly distributed throughout the body approximately one hour
post-injection. The signals accumulated in the tumor area quickly,
Pc-3 showed relatively higher PDTactivity and better tumor cell
selectivity in the cell-based MTT assay than did the other
conjugates. In addition, Pc-3 and Pc-5 showed better tumor
enrichment in the in vivo distribution assay than did the three
other conjugates. Thus, we selected Pc-3 as the photosensitizer for
further evaluating the tumor treatment potential by in vivo PDT
experiments. Human A431 tumors were first inoculated in the leg
Fig. 4. Comparative in vitro phototoxicity efficacy of Pc-1, Pc-2, Pc-3, Pc-4 and Pc-5 toward A549 (A), A431 (B), MCF-7 (C) and PC-3 (D) cells (n = 5, mean ꢃ SEM). Phototoxicity
efficacy after cells were treated with a series of concentrations of Pc-1, Pc-2, Pc-3, Pc-4 and Pc-5 under illumination for 10 min (laser wavelength of 660 nm and laser power
density of 40 mW/cm2).
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photosensitizers for tumor photodynamic therapy, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.04.025

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Fig. 6. Cell morphological changes observed by microscopy before and after PDT for A431 cells treated with Pc-1, Pc-2, Pc-3, Pc-4 and Pc-5.
Fig. 7. In vivo imagings of Pc-1-5 in xenograft mice bearing human A431 tumors. BALB/C nude mice bearing A431 tumors were intravenously injected with 25 nmol/mouse of
Pcs via the tail vein. Photographs were taken at 0, 1, 2, 4, 6, 9, 12, and 24 h after injection of Pcs. One representative mouse from each group (n = 4) is shown.
Please cite this article in press as: Q. Chen, et al., In vitro and in vivo evaluation of improved EGFR targeting peptide-conjugated phthalocyanine
photosensitizers for tumor photodynamic therapy, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.04.025

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Fig. 8. In vivo photodynamic therapeutic efficacy on mice bearing A431 tumor xenografts: 50 nmol/mouse of Pc-3 or PBS was injected intravenously, and irradiation was
performed at 150 mW/cm2 for 16 minutes (laser wavelength of 660 nm) two hours after injection (five mice in each group). All mice were treated with PDT only once. (A)
Representative images of mice injected with Pc-3 treated by 660-nm illumination. (B) Representative images of mice injected with PBS treated by 660-nm illumination. (C)
Tumor-growth curves of mice injected with Pc-3 and PBS. (D) Body weight of mice injected with Pc-3 and PBS on different days. Data shown are means ꢃ SEM (n = 5).
of BALB/c nude mice. When tumors reached 200 mm3, the mice
were randomly divided into two groups (n = 5 per group), with
each injected with 50 nmol/mouse of Pc-3 or PBS as a control,
followed by irradiation with a 660-nm laser (200 mW for 15 min)
2 h after injection. Tumor growth and weight were measured for
each mouse. As shown in Fig. 8A and B, the tumors in group 1
developed scabs, which gradually decreased in size and disap-
peared approximately 10 days after PDT. No tumors had regrown
40 days after the PDT treatment. One PDT treatment with Pc-3
appears to completely abolish tumors in the mouse xenograft
tumor model. Compared with those in group 1, the tumors grew
very rapidly in group 2, indicating that irradiation alone had no
influence on the inhibition of tumor growth. Fig. 8C shows tumor-
growth curves for mice receiving PDT treatment with Pc-3 or light
irradiation only. Fig. 8D shows the weights of the mice receiving
PDT treatment. The weight of the mice showed no obvious
differences among the groups, indicating that irradiation with Pc-3
had no distinct side effect on mouse weight and may thus meet the
necessary safety criteria for in vivo tumor treatment application.
In conclusion, we developed EGFR receptor-specific photo-
sensitizers based on peptide-conjugated highly water-soluble Pc
conjugates. Here, we report the synthesis, photophysical property
analysis, and in vitro and in vivo PDT studies of these Pc conjugates.
Among the conjugates, Pc-3 possessed better properties for
discriminating between healthy and tumor tissues and abolished
inoculated tumors in the subcutaneous xenograft tumor model,
making this approach a promising therapeutic agent for the
treatment of cancer.
Natural Science Foundation of China (No. 31527801) and the
National Natural Science Foundation of China (No. 81773293).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.cclet.2018.04.025.
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This research was supported by the National Key R&D Program
of China (No. 2017YFC1104400), the Major Program of the National
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photosensitizers for tumor photodynamic therapy, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.04.025

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Please cite this article in press as: Q. Chen, et al., In vitro and in vivo evaluation of improved EGFR targeting peptide-conjugated phthalocyanine
photosensitizers for tumor photodynamic therapy, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.04.025