Synthesis and biological evaluation of peptide-conjugated phthalocyanine photosensitizers with highly hydrophilic modifications

Article abstract of DOI:10.1039/c6ob00122j

The selectivity of targeted photosensitizer delivery is a constant challenge for photodynamic therapy (PDT). Herein, with the aim of optimizing the affinity, selectivity and activity of peptide-conjugated photosensitizers for PDT therapy and fluorescence

Full text of DOI:10.1039/c6ob00122j

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Synthesis and biological evaluation of peptide-conjugated
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phthalocyanine photosensitizer with highly hydrophilic modifications
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Fu Li,a,b† Qian Liu,b† Zhenzhen Liang,b Jin Wang,b Mingpei Pang,a,b Weiqiang
Huang,c,* Wenjie Wua,* and Zhangyong Hongb,*
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aCollege of Material Science and Chemical Engineering, Tianjin University of
Science and Technology, Tianjin 300457, P. R. China. E-mail: wwjie@tust.edu.cn;
Tel: +86 022 23502875.
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bState Key Laboratory of Medicinal Chemical Biology, College of Life Sciences,
Nankai University, Tianjin 300071, P. R. China. E-mail: hongzy@nankai.edu.cn; Tel:
+86 022 23498707.
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cKanghong Yaoyuan Biotech Co., Ltd, Tianjin 300192, P. R. China. E-mail:
hwq0324@hotmail.com; Tel: +86 022 23003610.
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‡Electronic Supplementary Information (ESI) available: [Synthetic procedure for
phthalonitriles and resin-bound peptide, HPLC chromatogram and ESI-HRMS of the
phthalocyanines]. See DOI:
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†Fu Li and Qian Liu contributed equally to this work.
Abstract：
The selectivity of targeted photosensitizer delivery is a constant challenge for
photodynamic therapy (PDT). Herein, with the aim of optimizing the affinity,

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selectivity and activity of peptide-conjugated photosensitizers for PDT therDaOpIy: 10a.1n03d9/C6OB00122J
fluorescence imaging, we designed a solid-phase strategy for the efficient synthesis of
peptide-phthalocyanine (Pc) conjugates with two types of highly hydrophilic
modifications to the Pc rings. The peptide conjugation clearly increased the
photodynamic efficacy and selectivity of Pcs against cancer cells with different
receptor expression levels. A highly hydrophilic modification to the Pc rings can
block non-specific interactions between the Pcs and biological molecules while
minimally influencing peptide ligand affinity with target receptors. Compared with
triethyleneglycol monomethyl ether group modification, the glycerol group
modification exhibited a stronger capability to decrease the Pc aggregation tendency
in aqueous medium, reduce non-specific binding and non-specific, light-induced
32 cytotoxicity towards cells with low receptor expression in vitro; increased targeting
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selectivity was observed in the in vivo distribution experiment via the obvious
reduction of background signals. Highly hydrophilic modifications to the Pc rings
may be very useful for improving targeting selectivity for PDT with the construction
of peptide-conjugated photosensitizers.
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Keywords: photodynamic therapy, photosensitizer, phthalocyanine, peptide conjugate
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1. Introduction
Photodynamic therapy (PDT) is a promising non-invasive modality for the treatment
of a variety of premalignant and malignant diseases.1 It utilizes the combined action

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of three individually harmless components, namely, a photosensitizer, light, and
molecular oxygen, to generate reactive oxygen species (ROS) leading to cellular and
44 tissue damage.2 Compared with traditional therapeutic methods, such as
45 chemotherapy, radiotherapy, and surgery, PDT has more control over the selective
46 destruction of malignant cells while sparing normal tissues and organ function;3 it also
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allows the toleration of repeated doses. In addition, photosensitizers are able to
provide intense fluorescence signals in tumor tissues, which thus makes them natural
candidates for the combination of fluorescence imaging and cancer therapy.4
Fluorescence imaging can offer superior sensitivity and real-time diagnosis in
51 monitoring and guiding therapy for cancer treatment.5
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However, conventional photosensitizers lack tumor selectivity, and considerable
damage occurs in healthy adjacent tissues. Because of this non-selectivity, the
fluorescence detection capability is also detrimentally influenced. More selective
photosensitizer targeting would be very useful but remains a major current challenge.
Some explorative efforts have been made in recent years to solve this problem;6 most
attempts have included the encapsulation of photosensitizers in colloidal carriers, such
as liposomes,7 polymeric micelles or nanoparticles8 to take advantage of the enhanced
permeability and retention (EPR) effect for tumor accumulation. Conjugation with
various tumor-specific vehicles, such as monoclonal antibodies,9 protein10 and peptide
61 ligands11 could provide other promising strategies. Among them, synthetic peptide
62 ligands are of particular interest for their excellent tumor penetration, low

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immunogenicity, and ease of conjugation to photosensitizers.12 Some
peptide-conjugated photosensitizers have been synthesized and showed improved
65 properties as a result of peptide conjugation.11
66 Phthalocyanine (Pc) is one of the most promising photosensitizer candidates for
67 PDT, and it has advantageous photophysical and photochemical properties.13 Pcs
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show strong absorption (extinction coefficient ε > 105 M−1 cm−1) at long wavelengths
(> 670 nm) and strong singlet oxygen generation abilities. Their spectral and
photophysical properties can be easily tuned by variation of substituents around the Pc
aromatic core or the central metal.14 It is possible to achieve improved targeting
capability of Pcs towards tumors by conjugating the Pcs with peptide ligands,
although only a few examples of Pcs functionalized with peptides have been
studied.15 To some extent, Pc conjugation with peptides can also alleviate the water
solubility and aggregation problems of Pcs; for most Pcs, extremely low solubility
and aggregation phenomena render them photodynamically inactive in aqueous
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In the literature, most peptide-Pc conjugates are constructed with non-modified
Pcs with peptide ligands.15 However, Pcs typically have super hydrophobic scaffolds
that involve multiple unsaturated double bonds linking multiple unsaturated rings.
These features lead non-modified Pcs to have high non-specific binding to many cells
and unwanted interactions with proteins and lipids in vivo, thus increasing the
non-specific background. In addition, the strong hydrophobicity of Pcs also has a

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serious influence on the binding function of the peptide ligands by destroying the
ligand selectivity. Therefore, in theory, hydrophilic Pc modification is necessary to
create ideal peptide-Pc conjugates12 by shielding the Pcs from non-specific binding
with biological molecules and maintaining specific binding between peptide ligands
and their targets. Not much work has been toward investigating the effects of
hydrophilic substitution on tumor selectivity and PDT outcome. How the substitution
pattern affects the properties of peptide-Pc conjugates, including affinity and
selectivity both in vitro and in vivo, is still an interesting topic.
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Herein, we want to construct peptide-Pc conjugates with highly hydrophilic
modifications on the Pc component with the aim of optimizing the affinity, selectivity
and activity of the Pc conjugates. Two types of non-ionic hydrophilic substituents, i.e.,
triethyleneglycol monomethyl ether substitution and glycol chain substitution, were
used for their lower interaction with constituents of biological fluids (e.g., plasma
proteins) and interfaces (e.g., cell membrane); these were used to increase Pc
hydrophilicity with the aim of comparing the effects of the hydrophilic substitutions
99 on the selectivity of the conjugates. D4 peptide LARLLT has a high affinity for
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epidermal growth factor (EGF),17 which is overexpressed on many human cancer cell
surfaces, including breast, lung, esophageal, head, and neck cancers;18 this peptide
was used as the model ligand for tumor targeting. A solid-phase strategy was adopted
to conjugate the peptide ligand with the hydrophilically substituted Pcs directly on
resin, which can offer the target conjugates without purification of the intermediates
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and may present one of the most convenient syntheses of peptide-Pc conjugates.
Here, we reported our synthesis and physical characterization of the peptide
conjugates of Pcs with two different hydrophilic substitutions. Their photodynamic
efficacy and intracellular localization against cancer cell lines with different
expression levels of EGF receptors were evaluated, and the in vivo distribution and
110 imaging capability were monitored via an in vivo imaging system.
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2. Results and discussion
2.1. Molecular design of water-soluble Pcs
The selectivity of photosensitizer targeted delivery is always a challenge in PDT. We
115 designed a solid-phase strategy for the efficient synthesis of peptide-Pc conjugates
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with highly hydrophilic substitutions on the Pc rings, with the aim of optimizing the
affinity, selectivity and activity of the conjugates for PDT therapy and fluorescence
imaging. Introduction of highly hydrophilic substitutions to the Pc rings can block the
non-specific interactions between Pcs and biological molecules, while minimally
influencing peptide ligand affinity with targets. For comparison purposes, two types
of hydrophilic substitutions, triethyleneglycol monomethyl ether groups and glycerol
groups, were adopted to reduce the hydrophobicity of the phthalocyanine ring. Herein,
asymmetrically substituted A3B-type Pcs with hydrophilic substitutions on the rings
and a single carboxylic acid were first designed to facilitate the formation of stable
amide bonds with amine-containing peptide ligands.
A
short peptide

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Lys-Ala-Arg-Leu-Leu-Thr, an EGF receptor-targeting peptide, was selected as the
ligand for the purpose of tumor targeting. The peptide was prepared according to the
standard 9-fluorenylmethoxycarbonyl (Fmoc) strategy in solid phase and was then
conjugated with the highly water-soluble A3B asymmetric Pcs on resin via amide
bond formation between the N-terminus of the peptide and the carboxylic group of the
Pcs. The syntheses of the peptide ligand and peptide-Pc conjugation were both carried
out in solid phase, which avoids the complex separation of intermediates. The final
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133 peptide-conjugated PcZn2 and PcZn4 were obtained directly after cleavage from the
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resin with trifluoroacetic acid (TFA). This new strategy to synthesize peptide-Pc
conjugates can produce the target peptide-Pc conjugates in a straightforward manner,
and modification of the Pc component can been easily carried out.
2.2. Synthesis of asymmetric tetra-substituted phthalocyanine PcZn1 and EGF
receptor-targeting peptide D4-conjugated phthalocyanine PcZn2
The asymmetrical A3B phthalocyanine PcZn1 with monosubstitution of a carboxyl
group was prepared via the statistical cyclotetramerization of a mixture of two
different phthalonitriles. After optimization of the feeding ratio of the two
143 phthalonitriles, the A3B-type PcZn1 could be obtained with quite a good yield after
144 silica gel purification when a 4-fold excess of monosubstituted phthalonitrile 2 versus
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phthalonitrile 1 was used. The ester of the Pc was subjected to hydrolyzation with
NaOH THF/methanol/water (2/5/1) and acidified with HCl to produce the Pc acid

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(PcZn1) at a yield of 16.9% in two steps via silica gel column purification. It DisOIw: 10o.1r0t3h9/C6OB00122J
148 noting that as the symmetric A4-type Pc and asymmetric A3B-type Pc were very close
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on the silica gel column, it was not easy to separate them, but PcZn1 can be simply
separated from the A4-type Pc after ester hydrolyzation.
The peptide D4 with side-chain protection was prepared on resin by using a Fmoc
solid-phase peptide synthesis (SPPS) protocol. After removing the Fmoc protecting
group with 20% piperidine in DMF, the N-terminus of the peptide 4 was coupled with
the carboxyl group of PcZn2 using 1-hydroxy-benzotriazole (HOBt) and
155 O-benzotriazol-1-yl-N,N,N',N'-tetra- methyluronium hexafluorophosphate (HBTU) as
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the activating agents. Then, the peptide-Pc conjugate was cleaved from the resin by
removing all protecting groups at the same time with 95% TFA (TFA/H2O/TIS =
95:2.5:2.5) to obtain the desired compound PcZn2 at a yield of 46% after HPLC
purification. The purity of the crude product cleaved from the resin was very good,
160 which greatly simplified the purification of the final product, PcZn2, by ether
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precipitation and HPLC purification. This method to produce the conjugated product
by coupling Pcs with peptide ligands directly on resin was very simple and did not
require complex separation and purification of intermediates.
2.3. Synthesis of asymmetric tetra-substituted phthalocyanine PcZn3 and EGF
receptor-targeting peptide D4-conjugated phthalocyanine PcZn4

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The asymmetrical A3B-type compound 7 was prepared via the same strategy with
PcZn1 by the reaction of phthalonitrile 1 and 6 followed by hydrolyzation, as shown
in scheme 2. For the acid lability of the isopropylidene group, we did not acidify the
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170 product after the hydrolyzation, but we purified the sodium salt of Pc 7 directly with a
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silica gel column. The conjugation of Pc 7 with the side-chain-protected peptide
ligand 4 on the resin failed to produce the target compound, although we tried many
types of conjugation conditions. Later we found that Pc 7 was not very stable in
174 strongly acidic conditions, such as high concentrations of TFA. A glycerol
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substitution on the -position of the Pc ring is reported to reduce the stability of Pcs
in strong acids. Therefore, we changed our synthesis to prepare the -substituted Pc
by the reaction of phthalonitrile 6 and 8 to form sodium salt of Pc 9 (24.1%) after
178 hydrolyzation of the ester group by the same strategy as was used with Pc 7. Pc 9 was
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acidified with 95% TFA (TFA/H2O/TIS = 95:2.5:2.5) to obtain PcZn3 (82.5%). At this
time, the Pc 9 was conjugated with the protected peptide ligand on resin to produce
the desired final product PcZn4 with a good yield (38.5%) after cleaving and
removing the protecting group with TFA.
2.4. Electronic absorption and photophysical properties
Absorption and fluorescence spectra in dimethylsulfoxide (DMSO)
186 UV-Vis absorption spectra of PcZn1~PcZn4 were measured in DMSO, as shown in
187 Fig. 1. All the Pcs have a Q absorbance band at approximately 680 nm, which is

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red-shifted by approximately 10 nm compared with non-substituted PcZn (~670 nm).
This can be explained by a reduction in the energy gap between the highest occupied
molecular orbitals (HOMO) and the lowest occupied molecular orbitals (LOMO),
191 resulting in a red-shift of the maximum absorption wavelength. The absorption
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spectra exhibit a good linear relationship with concentration (shown in SI, Fig. S2).
Fluorescence excitation and emission spectra in DMSO are shown in Fig. 1. All the
synthesized Pcs displayed a fluorescent excitation band at approximately 685 nm and
an emission band at approximately 695 nm. The emission spectra were similar to their
absorption spectra, and the emission spectra also have a good linear relationship with
concentration (shown in SI, Fig. S3).
Quantum yields, lifetimes and singlet oxygen quantum yields
200 The fluorescence quantum yields, lifetimes and singlet oxygen quantum yields of
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these compounds were determined according standard procedures and are shown in
Table 1. The absolute fluorescence quantum yields at room temperature were
determined on a PTI QM/TM/NIR system (Photon Technology International,
Birmingham, NJ, USA) with an integration sphere attachment (Labsphere, North
205 Sutton, NH, USA). A circular quartz cuvette for the solution sample is set in the
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integrating sphere. Fluorescence decay curves were measured on a PTI QM-4m
system (Photon Technology International, Birmingham, NJ, USA) with Felix32
software. The fluorescence quantum yields of PcZn1~PcZn4 were approximately

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0.10~0.14, and the lifetimes were approximately 3.0 ns. Singlet oxygen quantum yield
measurements were carried out using an experimental set-up described in literature
using 1, 3-diphenylisobenzofuran (DPBF) as a singlet oxygen chemical quencher and
ZnPc as a standard (singlet oxygen quantum yield of 0.67 in DMSO). The
213 PcZn1~PcZn4 compounds have singlet oxygen quantum yields of approximately
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0.50~0.51. It can be observed that all of these tetra-substituted Pcs are efficient singlet
oxygen generators that have singlet oxygen quantum yields comparable to that of
non-substituted ZnPc.
Aggregation study in aqueous systems
The aggregation of Pcs seriously hinders their application in PDT, especially in water
solution, as the aggregation greatly reduces fluorescence intensity and singlet oxygen
production. DMSO is known to prevent the aggregation of Pcs. The UV-Vis
absorption spectra of PcZn1~PcZn4 in DMSO all demonstrated desirable dispersive
behavior via sharp Q band absorption peaks and using the Lambert-Beer law in
concentrations from 1 μM to 10 μM (see SI, Fig. S2); similar results were shown by
their fluorescence spectra in different concentrations from 1 μM to 5 μM (see SI, Fig.
S3). However, DMSO is not an appropriate solvent to be used for studying biological
applications. Therefore, we adopted aqueous solutions to test the photochemical
properties of these Pcs to obtain more information for biological studies. The
absorbance and fluorescence spectra of PcZn1~PcZn4 in pure water, aqueous Tween

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80 solution (0.05% in water) and aqueous Triton X-100 solution (10% in watDeOr)I: 1w0.e10r3e9/C6OB00122J
determined, as shown in Fig. 2 and 3.
All of these four Pcs exhibited high solubility in pure water. From Fig. 2 and 3,
these four Pcs have quite good absorbance and fluorescence emission in pure water,
with moderate log (shown in Table 2). The peptide-conjugated PcZn2 and PcZn4
235 exhibited higher absorbance and fluorescence intensities than did their
236 non-conjugated counterparts. The introduction of peptide ligands alleviated, to some
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238 the triethyleneglycol monomethyl ether-substituted Pcs (i.e., PcZn1 & PcZn2) than in
239 the glycerol-substituted Pcs (i.e., PcZn3 & PcZn4). PcZn3 and PcZn4 exhibited higher
240 absorbance and fluorescence intensities than did PcZn1 and PcZn2. The glycerol chain
extent, the aggregation of the Pcs. However, the influence was much more obvious in
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substitution on the Pcs strongly increased the hydrophilicity and decreased the
aggregation tendency of the Pcs in aqueous media. However, to compare the data
from water with the data from DMSO, conjugation with peptide ligands was not able
to resolve completely the aggregation problems of Pcs in water, even though all of the
synthesized Pcs have high water solubility.
In biological systems, PDT agents could be dispersed to recover their function by
forming complexes with lipids, which are important components of cell and organelle
membranes, as long as they are sufficiently water soluble. The adhesion of PDT
agents to a cell membrane is important for them to be able to kill tumor cells.12
Therefore, pure water may be not an ideal system to mimic this biological

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environment. Here we mimicked the lipid environment of cell membranes by DaOdId: 1i0t.i1o03n9/C6OB00122J
of the detergent Tween 80 (0.05% in water) and Triton X-100 (10% in water) to the
water solution of these Pcs (concentration = 2 μM); we then assessed the dispersion of
the Pcs. From the absorbance spectra (Fig. 2), their Q band in aqueous Tween 80
solution is slightly broadened and slightly less intense than the spectrum in DMSO,
and their Q band in aqueous Triton X-100 solution was the same as in DMSO. From
their fluorescence emission spectra (Fig. 3), the fluorescence emission was also
almost recovered by the addition of 10% Triton X-100 and 0.05% Tween 80 in
aqueous solution. The recovery efficiency was higher in the glycerol-substituted Pcs
than in the triethyleneglycol monomethyl ether-substituted compounds. This result
was consistent with the absorbance spectra data. The results suggested that these Pcs
were all almost completely dispersed in aqueous Triton X-100 solution and only
exhibited a very small extent of aggregation in aqueous Tween 80 solution.
2.5. In vitro photodynamic activities tested by MTT
266 The photodynamic activities of the PcZn1, PcZn2, PcZn3, and PcZn4 compounds were
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evaluated against two cancer cell lines, namely, human epidermoid carcinoma (A431)
cells, which have high expression of epidermal growth factor (EGF), and human
breast adenocarcinoma cells (MCF-7), which have low expression of EGF. Fig. 4
shows the dose-dependent survival curves for these conjugates. All of these
compounds were essentially non-cytotoxic in the absence of light up to a

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272 concentration of 2.5 μM (PcZn1 and PcZn2) or 20 μM (PcZn3 and PcZn4). These
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results are quite similar with previous reports that have shown Pcs have good
biocompatibility with very low dark toxicity. Upon illumination with light (24 J/cm2),
non-peptide-conjugated PcZn1 and PcZn3 were still essentially nontoxic towards these
cells. With observations from the literature, the great reduction of the photodynamic
activity of the non-peptide-conjugated Pcs may be attributed to the highly hydrophilic
278 modification of the Pcs, which may greatly reduce their non-specific binding to cell
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membranes. The reduction of the non-specific photodynamic activity was even
obvious for the glycerol substitution, with more than 80% viability at 20 μM in the
presence of light. The great reduction of the non-specific, light-induced cytotoxicity
to cancer cells is very important for the enabling of peptide conjugate and ligand
targeting capability.
Upon illumination with the same dose of light (24 J/cm2), peptide-conjugated
PcZn2 and PcZn4 became very potent against A431 cancer cells with high EGF
receptor expression (IC50 of 0.15 and 0.65 μM, respectively). The presence of peptide
ligands on the peptide-conjugated PcZn2 and PcZn4 photosensitizers exhibited
targeting capability toward the EGF receptors expressed on the cell surface. The
conjugation of the Pcs with peptide ligands increased their PDT effects, possibly
through an increased uptake of the Pcs by cancer cells.
However, these two Pcs showed different activities towards the MCF-7 cells,
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PcZn4 showed much higher selectivity towards cells with high receptor exDpOreI:s10s.i1o03n9/C6OB00122J
levels and cells with low receptor expression levels than did PcZn2, by 32-fold and
2-fold, respectively. This result indicated that glycerol substitution may offer even
higher hydrophilicity and thus would have a higher capability of reducing
non-specific interactions with cancer cells than would triethyleneglycol monomethyl
298 ether substitution. High selectivity of PDT agents is very important for targeted PDT
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therapy.
2.9. Cellular uptake assays with confocal microscopy
To account for the different photodynamic efficacy and selectivity of PcZn2 and
PcZn4, we examined their cellular uptake by fluorescence microscopy (Fig. 5). A431
and MCF-7 cells plated on a coverslip were incubated with PcZn2 or PcZn4 for 4
hours in the dark, and the fluorescence signals of the Pcs were measured by CLSM at
690 nm (excitation at 610 nm).
It was observed that PcZn2 and PcZn4 showed different uptake efficiency and
selectivity towards A431 and MCF-7 cells. For the A431 cells, with high receptor
expression, the average fluorescence intensity was very strong for both compounds,
and increased fluorescence intensity was observed with a concentration increase from
311 10 μM to 20 μM. For the MCF-7 cells, with low receptor expression, PcZn4 showed
312 only very weak fluorescence signals in the MCF-7 cells when they were treated at 10
313 μM or 20 μM. However, PcZn2 still showed a strong uptake by the MCF-7 cells,

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especially at 20 μM, which demonstrated no selectivity for receptor expression level.
315 These results indicated that the peptide ligand could assist and increase the delivery of
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Pcs into cancer cells that overexpress EGF receptors. In addition, PcZn4 showed very
high selectivity for the receptor expression level of the cancer cells with reduced
non-specific binding to the cancer cells. To improve the targeting selectivity of
peptide conjugates, highly hydrophilic modifications, such as glycerol chain
substitutions, are much better than triethyleneglycol monomethyl ether substitutions.
These results are consistent with the MTT results.
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323 2.10. In vivo study
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We used an in vivo imaging system to monitor the distribution of PcZn1, PcZn2, PcZn3
325 and PcZn4 in tumor-bearing nude mice to evaluate the specific targeting capability of
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these conjugates towards EGF receptors. As shown in Fig. 6, for PcZn2 (Fig 6B), the
fluorescence signals were distributed throughout the mice 30 minutes after injection
and were observed to have slowly accumulated in the liver, spleen and kidneys 6
hours later. Then, the signals in the tumor remained consistent until 36 hours, while
the signals in the lungs and kidneys reduced obviously. At 48 hours, the signals in the
331 tumor site started to weaken. PcZn2 showed a tumor-targeting capability, but the
332 targeting was not very ideal, as it was contaminated with a high background. PcZn4
333 (Fig. 6D) behaved differently from PcZn2 inside the tumor-bearing mice. At 6 hours
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after injection of PcZn4, most of the fluorescence signals already showed strong
accumulation inside the tumor and liver; the signals then weakened in the liver while

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increasing in the tumor site. At 18 hours after the injection, the signals had mostly
accumulated in the tumor site and mostly disappeared in all the other organs. The
targeting capability of PcZn4 in the mice was clearly better than that of PcZn2. For the
non-peptide conjugated PcZn1 (Fig. 6A) and PcZn2 (Fig. 6C), the fluorescence signals
only have little accumulation at the tumor sites in 3 to 12 hours and quickly
eliminated in 18 hours. The peptide conjugation has significant effect on the targeting
of the PCs to the tumor site.
For further examination of the accumulation of the Pcs inside the organs, the mice
were euthanized 48 hours or 18 hours after the injection of PcZn2 or PcZn4, and their
organs (heart, liver, spleen, lungs, and tumor, as shown in Fig. 7A and 7B from left to
right) were collected for imaging. Both of the compounds showed strong
accumulation inside the tumor and only a small amount of accumulation inside the
348 liver. However, PcZn2 exhibited relatively higher accumulation in the kidneys. This
349 preliminary result indicated that the peptide LARLLT increased Pc targeting to the
350
351
352
353
354
355
356
tumor site, and the targeting selectivity could be increased by increasing the
hydrophilicity of the modification to the Pc rings. Highly hydrophilic substitutions on
the Pc rings can clearly reduce background signals.
3. Conclusion
We designed and synthesized peptide-Pc conjugates with two types of highly
hydrophilic modification to the Pc rings via a solid-phase strategy. The highly

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358
hydrophilic modification to the Pc rings can efficiently increase PDT selectivity by
blocking non-specific interactions between Pcs and cells. To compare with
359 triethyleneglycol monomethyl ether group-substituted PcZn2, the glycerol
360
361
362
363
364
365
366
group-substituted PcZn4 exhibited much lower non-specific binding and non-specific
light-induced cytotoxicity towards cells with low receptor expression in vitro (32-fold
and 2-fold selectivity, respectively), as well as much lower non-specific background
signals in the in vivo distribution experiment. Highly hydrophilic modification to the
Pc rings may be very useful for improving the targeting selectivity of PDT via the
construction of peptide-conjugated photosensitizers.
367 4. Experimental section
368
369
4.1. Chemicals and materials
Phthalonitrile 1, 2, 6 and 8 were prepared as described in the supporting information.
370 3-Nitrophthalonitrile, methyl 4-hydroxybenzoate, Triton X-100 and Tween 80 were
371
372
373
374
375
376
377
purchased from Alfa Aesar (Tianjin, China). 1, 3-Diphenylisobenzofuran (DPBF) was
purchased from J&K (Beijing, China). 4, 6-Diamidino-2-phenylindole (DAPI) was
purchased from Roche (Mannheim, Germany). Penicillin/streptomycin and fetal
bovine serum were purchased from HyClone (South Logan, UT, USA).
Trypsin-EDTA and high-glucose Dulbecco's modified Eagle medium (DMEM) for
cell culture were purchased from Gibco (Grand island, NY, USA). Deionized water
was obtained using the Barnstead NANOpure water purification system from Thermo

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379
Scientific (Waltham, MA, USA). All other chemicals and reagents used were of
analytical grade and obtained commercially unless stated otherwise.
380
381
382
4.2. Cells and animals
The A-431 and MCF-7 cells obtained from Saierbio (Tianjin, China) were cultured in
383 DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100
384
385
386
μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The
cells were routinely treated with 0.25% Trypsin-EDTA and were dissociated into a
single-cell suspension for future cell culture. BALB/c nude mice were purchased from
387 Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) and maintained
388
389
390
391
392
393
394
395
396
397
in a germ-free environment with free access to food and water. All animal procedures
were conducted under a protocol approved by the Institutional Animal Care and Use
Committee of Nankai University (Tianjin, China).
4.3. Instruments
1H spectra were recorded in DMSO-d6 solutions on a Bruker AV400 400 MHz
spectrometer. The mass spectra were recorded using a Varian 7.0T FTMS. Optical
spectra in the UV-visible region were recorded with a USA Cary 5000 using a 10-mm
path length cuvette at room temperature. Fluorescence measurements were performed
on a PTI QM/TM/NIR system (Photon Technology International, Birmingham, NJ,

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398
399
400
USA) equipped with a quartz cell (1 cm × 1 cm).
4.4. Synthesis experiments
401 Synthesis of PcZn1. Phthalonitrile 1 (27.8 mg, 0.10 mmol), phthalonitrile 2 (100 mg,
402
403
404
405
406
0.34 mmol), anhydrous Zn(OAc)2 (31.0 mg, 0.17 mmol) and DBU (40 μL) were
mixed in n-hexyl alcohol (1.0 mL) in a sealed tube and stirred at 160 °C for 4 h. After
cooling, the crude product was poured into 10 mL of petroleum ether, and the
collected green solid was purified through a silica gel column using
dichloromethane:methanol (10:1) as an eluent to produce the green Pc contaminated
407 with the symmetric A4-type Pc. The mixture was then subjected to hydrolyzation with
408
409
410
411
412
NaOH (200 mg) dissolved in THF/water/methanol (2:1:5, 8 mL). The mixture was
stirred at 30 °C for 30 minutes and then acidified with 5 M HCl to pH 2-3. The
resulting green precipitate was collected through centrifugation and purified with a
silica gel column eluted with dichloromethane:methanol (10:1) to produce a green
powder (PcZn1, 20.3 mg, 16.9% in two steps). HRMS (ESI): calcd for C60H62N8O15Zn
413 [M + H]+ 1199.3699, found 1199.3692. 1H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J =
414
415
416
118.7 Hz, 5H), 8.14 (dd, J = 135.6, 86.1 Hz, 7H), 7.47 (s, 5H), 5.76 (s, 4H), 4.57 (s,
5H), 4.13 (d, J = 46.7 Hz, 5H), 3.93 – 3.59 (m, 20H), 3.50 (s, 6H), and 3.27 (s, 5H).
417 Synthesis of PcZn2. Peptidyl resin (100 mg, 50 μmol) was suspended in DMF (5 mL)
418 for 1 h before 20% piperidine in DMF (5 mL) was added to remove Fmoc protecting

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419
420
groups and DMF was used for washing (3 × 5 mL). The peptidyl resin 4 reDcOeiI:v1e0.d103a9/C6OB00122J
mixture of PcZn1 (60 mg, 50 μmol), HBTU (100 mg, 250 μmol), and DIEA (30 μL,
421 250 μmol) in 5 mL of DMF. The mixture was shaken overnight at room temperature
422
423
424
425
426
427
428
429
and was washed with DMF and dichloromethane before being dried under vacuum.
Cleavage and de-protection were performed by treatment of the dried resin with 200
μL of cleavage solution (TFA/Tis/water = 95:2.5:2.5) for 40 minutes at room
temperature. The product was precipitated and washed four times with ether (2 mL)
and dried under reduced pressure to produce PcZn2 (43.2 mg, 46%) after HPLC
purification. HRMS (ESI): calcd for C91H121N19O21Zn [M + H]+ 1880.8349, found
1880.8342. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 2H), 8.31 (s, 3H), 8.24 (s, 2H),
8.03 (s, 1H), 7.88 (s, 4H), 7.73 (s, 1H), 7.59 (s, 1H), 7.25 (s, 3H), 4.18 (d, J = 8.0 Hz,
430 35H), 3.89 (s, 2H), 3.51 (d, J = 14.0 Hz, 2H), 3.20 (d, J = 5.8 Hz, 3H), 2.86 (d, J =
431
432
433
434
435
436
437
438
439
13.9 Hz, 3H), 1.78 (s, 5H), 1.61 (d, J = 6.7 Hz, 16H), 1.47 (s, 4H), 1.35 (d, J = 7.0 Hz,
6H), 1.21 (d, J = 14.0 Hz, 2H), 1.10 (d, J = 6.2 Hz, 6H), and 1.03 – 0.77 (m, 20H).
The purity of the product was assessed using HPLC.
Synthesis of PcZn3. Phthalonitrile 6 (111.6 mg, 0.432 mmol), phthalonitrile 8 (30 mg,
0.108 mmol), anhydrous Zn(OAc)2 (40 mg, 0.216 mmol) and DBU (33 μL) were
mixed in n-hexyl alcohol (1.0 mL) in a sealed tube and stirred at 160 °C for 4 h. After
cooling, the crude product was separated by being pour into 10 mL of petroleum ether,
and the collected green solid was purified over a silica gel column using ethyl

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440
441
442
acetate:petroleum:methanol = 20:20:1 as an eluent to produce the green Pc, which
was contaminated with the symmetric A4-type Pc. The mixture was then subjected to
hydrolyzation with NaOH (200 mg) dissolved in THF/water/methanol (2:1:5, 8 mL).
443 The mixture was stirred at 30 °C for 30 minutes and then extracted with
444
445
446
dichloromethane (3 × 5 mL). The organic layer was dried over NaSO4 and
concentrated. The resulted green solid was purified with a silica gel column eluted
with dichloromethane:methanol (10:1) to produce Pc 9 (green powder, 26.5 mg, 24.1%
447 in two steps). HRMS (ESI): calcd for C57H50N8O12Zn 1102.2840, found 1102.2838.
448
449
450
451
1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 9.46 – 9.16 (m, 2H), 8.88 (s, 2H), 8.41
(s, 5H), 7.85 (t, J = 27.9 Hz, 2H), 7.33 (dd, J = 79.4, 34.3 Hz, 3H), 6.98 (dd, J = 36.9,
13.7 Hz, 2H), 4.24 – 4.18 (m, 2H), 3.76 (s, 13H) and 1.92 – 1.24 (m, 18H).
Pc 9 (25 mg) was dissolved in a solution of TFA/H2O = 95/5 (200 μL) and shaken for
452 40 min at room temperature. The addition of cold Et2O and repeated washing with
453 Et2O yielded solid PcZn3 (18.4 mg, 82.5%). HRMS (ESI): calcd for C48H39N8O12Zn
454 [M + H]+ 983.1973, found 983.1948. 1H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H),
455
456
457
458
459
460
8.92 (s, 3H), 8.75 (s, 1H), 8.66 – 8.22 (m, 4H), 7.99 (d, J = 64.2 Hz, 3H), 7.64 (d, J =
46.1 Hz, 4H), 7.50 – 7.10 (m, 2H), 4.71 (s, 4H), 4.58 (s, 6H), 4.34 (s, 5H), 3.93 (s, 3H)
and 3.52 (dd, J = 19.0, 16.3 Hz, 3H).
Synthesis of PcZn4. Peptidyl resin (100 mg, 50 μmol), after removal of the Fmoc
protecting groups, was mixed with Pc 9 (55 mg, 50 μmol), HBTU (100 mg, 250

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461
462
463
464
465
466
467
μmol), and DIEA (30 μL, 250 μmol) in 5 mL of DMF. The mixture was shaken
overnight at room temperature and washed with DMF and dichloromethane before
being dried under vacuum. Cleavage and de-protection were performed by treatment
of the dried resin with 200 μL of cleavage solution (TFA/Tis/water = 95:2.5:2.5) for
40 minutes at room temperature. The product was precipitated and washed four times
with ether (2 mL) and dried under reduced pressure to produce PcZn4 (32 mg, 38.5%)
after HPLC purification. HRMS (ESI): calcd for C79H97N19O18Zn [M+H]+ 1664.6623,
1
468 found 1664.6620. H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 6.8 Hz, 3H), 8.24 –
469
470
471
472
473
474
475
476
477
478
479
480
481
8.18 (m, 3H), 7.90 (d, J = 8.0 Hz, 2H), 7.76 (s, 2H), 7.59 (s, 2H), 7.49 (d, J = 8.4 Hz,
2H), 7.13 (d, J = 8.8 Hz, 3H), 4.40 – 4.31 (m, 6H), 3.86 (s, 5H), 3.09 (s, 4H), 2.82 –
2.72 (m, 5H), 1.65 (d, J = 16.0 Hz, 5H), 1.58 – 1.42 (m, 16H), 1.38 (d, J = 10.8 Hz,
4H), 1.25 (d, J = 7.0 Hz, 6H), 1.09 (dd, J = 15.9, 8.9 Hz, 2H), 1.00 (d, J = 6.2 Hz, 6H),
0.92 – 0.79 (m, 20H). The purity of the product was assessed using HPLC.
4.5. Photophysical and photochemical properties
UV-Vis and fluorescence spectra
The UV-Vis spectra were measured using a USA Cary 5000 with a 1-cm path length
cuvette. The concentration of Pcs was 10 μM in the solvents, including DMSO, pure
water, 10% Triton X-100 aqueous solution and 0.05% Tween 80 aqueous solution.
Baselines and spectra were recorded at room temperature in spectrum ranging from
500-900 nm with a 1-nm resolution and a 600-nm / min scan rate.

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484
485
486
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488
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490
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492
The fluorescence emission spectra were recorded in the wavelength range of D6O0I0: 1-09.100309/C6OB00122J
nm upon excitation at 680 nm, and the fluorescence excitation spectra were recorded
in the wavelength range of 500-900 nm upon emission at 695 nm. The concentration
of Pcs was 2 μM in the solvents, including DMSO, pure water, 10% Triton X-100
aqueous solution and 0.05% Tween 80 aqueous solution. The slit widths were 1 and 2
nm for excitation and emission, respectively.
Natural radiative life times
Fluorescence decay curves were measured using a PTI QM-4m system (Photon
Technology International, NJ) with Felix32 software. The photomultiplier tube (PMT)
voltage was set to 1074 V. The decayed curves show Pc emission at 695 nm after
493 excitation by a high-resolution laser (N2 laser at 337 nm tuned by laser dye PLD 665)
494
495
496
497
498
at 665 nm.
Quantum yield
The absolute photoluminescence quantum yield (QY) of the solutions at room
temperature was determined using a PTI QM/TM/NIR system (Photon Technology
499 International, Birmingham, NJ, USA) with an integration sphere attachment
500
501
502
(Labsphere, North Sutton, NH, USA). A circular quartz cuvette for the solution
samples was set in the integrating sphere. The absolute photoluminescence QY is
given by:

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Lsample
PN(em)
PN(abs)

QY 

Ereference
sample

 E
503
504
where PN (abs) and PN (em) represent the number of photons absorbed by the sample
and emitted from the sample, respectively. Lsample represents the photoluminescence
intensity of the sample (in DMSO solution), and Ereference and Esample represent the
excitation light intensities of the reference and the sample, respectively. Pure DMSO
was used as a reference.
505
506
507
508
509
510
511
512
513
514
515
Singlet oxygen quantum yields
Singlet oxygen quantum yield measurements were performed using an experimental
set-up described in the literature. 1,3-Diphenylisobenzofuran (DPBF) was used as a
singlet oxygen chemical quencher, and ZnPc was used as a standard with a quantum
yield of 0.67 in DMSO. The concentration of each compound was 10 μM in DMSO,
the DPBF concentration was 25 μM in DMSO, and a light intensity of 6.0 × 1015
516 photons s-1 cm-2 was used for the singlet oxygen quantum yield determinations. The
517
518
519
reduction of DPBF concentration was monitored at 417 nm for the degradation of the
solutions during irradiation. The singlet oxygen quantum yield was calculated
according to the following formula:
Std
r
(1 10 A
)
)
Pc
DPBF
685
520
Pc  Std
Pc
685
Std
DPBF
r
(1 10 A
521
522
where ΦΔ represents the singlet oxygen quantum yield (ΦΔ = 0.67 for the standard
Pc
Pc
DPBF
Std
DPBF
represent the DPBF photobleaching rates, 1-10-A
685
ZnPc in DMSO), r
and r

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Std
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Pc
Std
685
523
524
and 1-10-A represent the fraction of absorbed light, and A685 and A685 represent the
absorbance at the irradiation wavelength of the Pcs and the standard ZnPc,
respectively.
525
526
527
4.6. In vitro PDT activity assays
528
A431 and MCF-7 cells were seeded in each well of 96-well plates at a density of
529 1×105 cells/mL of culture medium and incubated overnight. Then, the cells were
530
531
washed with phosphate-buffered saline (PBS), and various concentrations of Pcs
dissolved in the culture medium were added into two plates for 4 h at 37 °C under 5%
532 CO2. One plate was irradiated with a red light of 660 nm for 10 min, while the other
533
534
535
536
served as a dark control. After 24 hours of incubation, cell viability was measured
using a BioTek microplate reader using the MTT colorimetric assay measured at 490
nm.
537
538
539
540
541
542
543
544
4.7. Cellular uptake assays
Cells (5×104) in complete medium (500 μL) were plated on a coverslip and cultured
overnight. After removal of the medium, the cells were incubated with 10 μM or 20
μM Pcs for 4 h in the dark and washed with PBS buffer three times to eliminate any
unbound Pcs. Then, 300 μL of fixing solution (4% paraformaldehyde) and 300 μL of
DAPI solution (1 μg mL−1) were subsequently incubated with the cells at room
temperature for 15 min and 5 min, respectively. Then, the cells were washed with
PBS buffer and imaged with a confocal laser scanning microscope (TCS SP8, Leica,

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546
547
548
549
550
Wetzlar, Germany).
4.8. In vivo imaging
Female BALB/c nude mice (7 weeks of age) received subcutaneous implants
consisting of 3×106 A431 cells suspended in 100 μL of PBS in their back. Once the
tumors grew to 50 mm3 (approximately 2 weeks post-tumor inoculation), PcZn1,
551 PcZn2, PcZn3 or PcZn4 dissolved in PBS was injected via the tail vein at a dose of 50
552 nmol per mouse, while the mice of the control group were injected with the same
553 volume of PBS. The in vivo fluorescence analysis was performed before and after the
554 injection at various time points using a Xenogen in vivo imaging system with a Cy5.5
555
556
filter (excitation 615 to 665 nm, emission 695 to 770 nm). The mice were euthanized
at 12 h or 48 h post-injection, and major organs including the heart, liver, spleen,
557 lungs, and kidneys as well as the tumor were collected; fluorescence images were then
558
559
acquired.
560 Acknowledgements
561
562
563
This work was supported by the National Natural Science Foundation of China (No.
31270926) and the Natural Science Foundation of Tianjin (No. 13JCZDJC28300).
564 References
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