Synthesis and biological evaluation of phthalocyanine-peptide conjugate for EGFR-targeted photodynamic therapy and bioimaging

Accepted Manuscript Synthesis and biological evaluation of phthalocyanine-peptide conjugate for EGFRtargeted photodynamic therapy and bioimaging Ligang Yu, Qiong Wang, Roy C.-H. Wong, Shirui Zhao, Dennis K.P. Ng, Pui-Chi Lo PII:

S0143-7208(18)32415-X

DOI:

https://doi.org/10.1016/j.dyepig.2018.11.055

Reference:

DYPI 7199

To appear in:

Dyes and Pigments

Received Date: 1 November 2018 Revised Date:

27 November 2018

Accepted Date: 28 November 2018

Please cite this article as: Yu L, Wang Q, Wong RC-H, Zhao S, Ng DKP, Lo P-C, Synthesis and biological evaluation of phthalocyanine-peptide conjugate for EGFR-targeted photodynamic therapy and bioimaging, Dyes and Pigments (2018), doi: https://doi.org/10.1016/j.dyepig.2018.11.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Synthesis and biological evaluation of phthalocyanine-peptide conjugate for EGFR-targeted photodynamic therapy and bioimaging

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Ligang Yua, Qiong Wanga, Roy C.-H. Wongb, Shirui Zhaob, Dennis K. P. Ngb, PuiChi Loa,*

a

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Department of Biomedical Sciences, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

b

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Department of Chemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, China

ABSTRACT: To improve the biocompatibility and tumor selectivity, we employed an epidermal growth factor receptor (EGFR) binding peptide (namely GE11, with a sequence of Tyr-His-Trp-

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Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile) as a tumor directing vector for the delivery of zinc(II) phthalocyanine for targeted photodynamic therapy and bioimaging. The photophysical properties, cellular uptake, in vitro cytotoxicity, and in vivo biodistribution of this phthalocyanine-peptide

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conjugate (namely Pc-GE11) have been evaluated. Pc-GE11 exhibited higher cellular uptake on EGFR-overexpressing epidermoid carcinoma A431 cells when compared with that on human

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breast adenocarcinoma MCF7 cells (low EGFR expression). Moreover, pretreatment of A431 cells with GE11 peptide inhibited the cellular uptake of Pc-GE11 significantly and it exhibited exclusive light-activated cytotoxicity toward A431 cells. Furthermore, Pc-GE11 showed much higher tumor accumulation than the non-targeted control compound containing a random peptide sequence (Tyr-Trp-Gly-Pro-Asn-Ile-His-Tyr-Tyr-Thr-Gln-Val) after intravenous administration in A431-tumor bearing mice, indicating the potential application of this GE11 peptideconjugated photosensitizer for targeted photodynamic therapy and bioimaging. 1

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1. Introduction Photodynamic therapy (PDT) has long been used for the treatment of cancers and various

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diseases. It involves the combined action of a photosensitizer, molecular oxygen, and an appropriate wavelength of light to generate reactive oxygen species (ROS) for the eradication of cancer cells and tissues [1-3]. Currently, there are only few clinically approved PDT agents,

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including porfimer sodium, 5-aminolevulinic acid, temoporfin, verteporfin, talaporfrin, and padeliporfin [3]. However, the limited tumor selectivity and poor pharmacokinetics of these PDT

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agents have become major obstacles for their clinical application. To enhance the selectivity of PDT, “smart” photosensitizers have been developed, in which their photosensitizing properties are quenched through Foster resonance energy transfer or self-quenching mechanisms until they interact with specific cancer-associated stimuli [4-5]. Alternatively, photosensitizers can be conjugated with tumor-specific vectors, such as antibodies, peptides, carbohydrates, or other

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small biomolecules, to enhance the tumor-targeting property [6-9]. Synthetic peptides have been widely used as the tumor-targeting ligands for cancer treatment and imaging [10-13]. With the

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aid of phage-display peptide library, a large number of peptide ligands have been identified which can specifically bind to different cancer cells, cancer-associated cell surface receptors, and

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tumor vasculatures [14]. In addition to the high specificity, peptide-based drugs also have other advantages such as the ease of preparation and modification, non-immunogenicity, high tissue permeability, and rapid clearance from the body [15]. This approach has been widely used for the delivery of various agents such as chemotherapeutic drugs [16-18], radiopharmaceuticals [19-20], antisense oligonucleotides [21-23], and therapeutic nanoparticles [13, 24-25]. A substantial number of peptide-conjugated photosensitizers have been synthesized and evaluated for targeted PDT [26]. Peptide-conjugated photosensitizers usually exhibit less 2

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aggregation tendency and improved solubility in aqueous solution, hence enhancing the photosensitizing properties and therapeutic efficacy. Tumor homing peptides are one of the most interesting series which can target the receptors expressed on tumor tissues. Rahimipour et al.

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have conjugated protoporphyrin IX (PpIX) with a gonadotropin-releasing hormone (GnRH) agonist or antagonist to improve its selectivity toward cancerous cells that express GnRH receptors [27]. Another phthalocyanines conjugated with GnRH peptides have also been

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developed and they exhibited good antitumor efficacy on a GnRH receptors expressing murine breast cancer 4-T1 bearing mice [28]. A chlorin-type photosensitizer conjugated with the

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heptapeptide has also been developed to target the vascular endothelial growth factor (VEGF) co-receptor neuropilin-1, both in vitro and in vivo [29-30]. Several porphyrin, chlorin, and phthalocyanine-based photosensitizers conjugated with a linear or cyclic RGD motif have also been prepared and studied for their in vitro and in vivo selectivity and PDT efficacy toward

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αvβ3-integrin expressing human endothelial cells [31-37]. Recently, a ruthenium-somatostatin photosensitizer has also been prepared for targeted PDT. It showed rapid cellular uptake by receptor-mediated endocytosis and potent photocytotoxicity on human lung cancer cells A549

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expressing somatostatin receptors [38].

Among all these tumor-related receptors on the tumor cells, epidermal growth factor

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receptors (EGFR) are particularly interested as they play essential roles in both normal physiological and cancerous conditions. EGFR are expressed in a variety of human tumors, including lung, colon, pancreas, breast, ovary, bladder, kidney, head and neck, and gliomas, therefore they serve as promising therapeutic targets [39-40]. Several anti-EGFR therapeutic agents, including monoclonal antibodies (e.g. cetuximab and panitumumab) and tyrosine kinase inhibitors (e.g. gefitinib, erlotinib, and lapatinib) have been developed [41]. Recently, EGFR-

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targeted GE11 peptide has been identified by phage display screening. It contains 12 amino acids with a peptide sequence of Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile and it can bind to EGFR competitively with EGF and mediate internalization to EGFR-expressing cancer cells [42].

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The GE11 peptide has been conjugated with radiolabeled and fluorescent probes [43-44], and several drug delivery systems to deliver doxorubicin, paclitaxel, gemcitabine, aminoflavone, and siRNA for the EGFR-targeted diagnosis and treatment of cancers [45-50]. More recently, several peptide-decorated

nano-photosensitizing

systems

encapsulating

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GE11

silicon(IV)

phthalocyanines and chlorin e6 have been explored for EGFR-targeted PDT [51-53]. In addition,

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chlorin e4 and zinc(II) phthalocyanine conjugated with GE11 peptide have been prepared, showing improved selectivity but weak absorption in the red region and only moderate PDT efficacy [54-56]. Therefore, there is still room for the development of photosensitizer-GE11 peptide conjugate for EGFR-targeted PDT with potent therapeutic efficacy. As an extension of

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our exploration of peptide-based photosensitizers [34, 57], we report herein the synthesis, characterization, photophysical properties, and in vitro photodynamic activity of a zinc(II) phthalocyanine conjugated with GE11 peptide (Pc-GE11). The in vivo biodistribution of this

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conjugate has also been examined on tumor-bearing nude mice.

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2. Results and discussion

2.1 Synthesis and characterization Scheme 1 shows the synthetic route of the Pc-GE11. The side-chain protected peptide resin was synthesized according to the standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocol [58] with commercially available N-α-Fmoc-protected amino acids and Sieber amide resin as the solid support. The 1,4-bis(triethylene glycol)-substituted

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carboxyl zinc(II) phthalocyanine (Pc-COOH), which exhibits a red-shifted absorption, enhanced 1

O2 generation, and reduced aggregation tendency as compared with unsubstituted zinc(II)

phthalocyanine (ZnPc), was synthesized according to the literature procedure [57]. Firstly, the

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Fmoc protecting group of the tyrosine was removed by 20% of piperidine in DMF, then the peptide-resin was reacted with Pc-COOH in the presence of 1-hydroxybenzotriazole (HOBt), O(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU), and N,N-

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diisopropylethylamine (DIPEA). The dark-green resin obtained was treated with a solution containing trifluoroacetic acid (TFA) (88%), phenol (5%), triisopropylsilane (TIS) (2%) and H2O

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(5%) to remove all the protecting groups on the peptide sequence and cleave the peptide from the resin. The crude Pc-GE11 conjugate was purified by semi-preparative HPLC. The control compound (Pc-rGE11), which contains a random sequence peptide (Tyr-Trp-Gly-Pro-Asn-IleHis-Tyr-Tyr-Thr-Gln-Val), was also prepared by the same method. Both Pc-GE11 and Pc-

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rGE11 were characterized by analytical HPLC and ESI mass spectrometry (Fig. S1-S4 in the Supporting Information). The purities of Pc-GE11 and Pc-rGE11 were found to be more than

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97% as determined by analytical high-performance liquid chromatography (HPLC).

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N

N

N

N

Zn

N

2. Pc-COOH, HOBt, HBTU, DIPEA

O

O

O

N

N

N

O

O

O

Pc-COOH

N

Zn

N

N

N

N

N

N

N

N

Zn

N

N

N

N

O

O

O

O

O

O

O

O

O

O

O

O

N N N

O C HN

N N

OH C O

TFA/phenol/TIS/H 2O (88:5:2:5)

O

O

N

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N

N

O

O

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N

O

O

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1. 20% piperidine in DMF

N N N

O C HN

Pc-GE11

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Scheme 1. Synthetic route of zinc(II) phthalcyanine conjugated with GE11 peptide (Pc-GE11).

2.2 Spectroscopic and photosensitizing properties

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The electronic absorption and fluorescence spectra of Pc-GE11 were examined in DMF

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(Fig. 1a) and compared with Pc-COOH. It can be seen that Pc-GE11 shows a Soret band at 338 nm, a vibronic band at 623 nm, and a Q-band at 691 nm. The Q-band absorbance of Pc-GE11 was weaker than that of Pc-COOH, suggesting certain degree of aggregation behavior of PcGE11 after the substitution of the GE11 peptide. Upon excitation at 610 nm, the fluorescence of Pc-GE11 at 708 nm was observed and it was also slightly weaker than that of Pc-COOH (Fig. 1b), which may be attributed to the aggregation or the quenching effect of the tryptophancontaining GE11 peptide [54-55]. The absorption and photophysical data are summarized in 6

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Table 1. To mimic the biological environment, we also measured their absorption, fluorescence emission and singlet oxygen (1O2) generation in phosphate buffered saline (PBS) being formulated with 0.05% Tween 80 (v/v). As shown in Fig. 2, both compounds exhibited very

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similar absorption and fluorescence spectra after formulated with surfactant in aqueous solution, suggesting that the GE11 peptide conjugation does not affect the core of the phthalocyanine and the quenching effect by the tryptophan moiety is not significant in this condition. The 1O2

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generation efficiency was also determined in PBS using 1,3-diphenylisobenzofuran (DPBF) as the scavenger. The concentration of the quencher was monitored spectroscopically at 415 nm

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during irradiation. As shown in Fig. S5 in the Supporting Information, both Pc-GE11 and PcCOOH could generate 1O2 efficiently and the efficiency is comparable to each other. It further proves that conjugation of GE11 peptide to the zinc(II) phthalocyanine do not interfere the

0.20 0.16

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0.12 0.08

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Absorbance

(b)

Pc-COOH Pc-GE11

0.04 0.00

300

400

500

600

700

800

Wavelength (nm)

Fluorescence Intensity (a.u.)

(a)

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photosensitizing properties.

2000

Pc-COOH Pc-GE11

1600 1200 800 400 0 640

660

680

700

720

740

760

780

800

Wavelength (nm)

Fig. 1. (a) Electronic absorption and (b) fluorescence spectra (excitation at 610 nm) of PcCOOH and Pc-GE11 in DMF (both at 1 µM).

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0.12

Absorbance

0.10 0.08 0.06 0.04 0.02 0.00 300

400

500

600

700

800

1000 Pc-COOH Pc-GE11

800

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(b)

Pc-COOH Pc-GE11

600 400 200 0 640

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0.14

Fluorescence Intensity (a.u.)

(a)

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680

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Wavelength (nm)

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Fig. 2. (a) Electronic absorption and (b) fluorescence spectra (excitation at 610 nm) of PcCOOH and Pc-GE11 in PBS (pH 7.4) formulated with 0.05% Tween 80 (both at 1 µM).

Table 1. Electronic absorption and photophysical data for Pc-GE11 and Pc-COOH in DMF.

Pc-COOH Pc-GE11

Φ Fb

Φ△c

688 (5.24)

705

0.14

0.63

691 (5.12)

708

0.10

0.48

Excited at 610 nm. b Relative to ZnPc in DMF as the reference (ΦF = 0.28). cRelative to ZnPc as

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a

λem (nm)a

λmax (nm) (log ε)

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Compound

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the reference (Φ∆ = 0.56 in DMF).

2.3 In vitro studies

To examine the targeting property of the Pc-GE11 conjugate, we examined the cellular uptake of this conjugate toward human epidermoid carcinoma A431 cells (with high expression of EGFR) and human breast carcinoma MCF7 cells (with low expression of EGFR) by flow cytometry. It is clear that the cellular uptake of Pc-GE11 is much higher on A431 cells when 8

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compared with MCF7 cells as shown in Fig. 3a. In addition, upon the addition of excess GE11 peptide (100 or 400 µM), the cellular uptake of Pc-GE11 was significantly inhibited on A431 cells and the trend is concentration-dependent. Pretreatment of 100 µM or 400 µM of GE11

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peptide on A431 cells could block 25 % or 50% of the cellular uptake of Pc-GE11, respectively (Fig. 3b). The competitive study clearly demonstrated the EGFR-mediated pathway for the internalization of Pc-GE11. In addition, we compared the cellular uptake of Pc-GE11 with the

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control substituted with scrambled peptide sequence Pc-rGE11. It is obvious that the cellular uptake of Pc-rGE11 was significantly lower than Pc-GE11 on A431 cells. The flow cytometric

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results are summarized in Fig. 4. Apart from using the flow cytometry, we also examined the cellular uptake of Pc-GE11 on A431 cells by confocal microscopy. As shown in Fig. 5, there is a

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strong intracellular fluorescence in the cytoplasm of the cells after treated with Pc-GE11 for 4 h.

Fig. 3. (a) Flow cytometric analysis of A431 and MCF7 cells after treated with Pc-GE11 (4 µM) for 4 h. (b) Flow cytometric analysis of A431 cells after pretreated with free GE11 peptide (100 or 400 µM) for 2 h, followed by incubation with Pc-GE11 (4 µM) for 4 h.

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Fig. 4. Comparison on the intracellular fluorescence intensity of Pc-GE11 and Pc-rGE11 in A431 and MCF7 cells monitored by flow cytometry. Data are expressed as the mean±standard

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deviation of three independent experiments.

Fig. 5. Confocal microscopic images of A431 cells after treated with Pc-GE11 (4 µM) for 4 h.

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The dark- and photo-cytotoxicity of Pc-GE11 was examined on A431 cells. Fig. 6. shows the dose-dependent survival curves both in the absence and presence of light. It is clear that PcGE11 is non-cytotoxic in dark at the tested concentrations, while it exhibits good cytotoxicity

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upon illumination. The IC50, which defined as the concentration of the dye required to kill 50% of the cells, was found to be 14 µM. The light-activated cytotoxicity makes Pc-GE11 a

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promising PDT agent.

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Cell Viability (%)

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in dark with light

80 60 40

0

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0

4

8

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16

20

24

28

32

[Pc-GE11] (µM)

Fig. 6. Cytotoxic effects of Pc-GE11 on A431 cells in the absence and presence of light (λ > 610

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nm, 18 mW cm-2, 32 J cm-2). Data are expressed as mean value ± standard error of the mean

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(S.E.M.) of three independent experiments, each performed in quintuplicate.

2.4 In vivo bio-distribution To study the in vivo tumor targeting ability of Pc-GE11, nude mice bearing A431 tumor were intravenously injected with Pc-GE11 (1 nmol per gram of mouse) and its bio-distribution was monitored for 72 h continuously as reflected by the measurement of the whole body fluorescence intensity. The non-targeted compound Pc-rGE11 was also used as the control. Both Pc-GE11 11

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and Pc-rGE11 spread over the whole body after injection for 4 h and the fluorescence intensity in the mice decreased significantly after 10 h post-injection. The Pc-GE11 showed much better tumor-retention property after 24 h post-injection. Moreover, bright fluorescence of Pc-GE11

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was also observed in the tumor region during 24-72 h post-injection. However, the fluorescence intensity of Pc-rGE11 at the tumor site was not significant throughout the study. To evaluate the bio-distribution of both compounds in tumor and different organs, the mice were sacrificed 3

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days after intravenous injection. The tumor and organs (kidney, heart, liver, lung, spleen and skin) were harvested and their relative fluorescence intensities were then measured. As shown in Fig. 8,

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the fluorescence intensities of Pc-GE11 in different organs were similar with Pc-rGE11, however, the fluorescence intensity of Pc-GE11 in tumor was much higher than that of PcrGE11, further proving the elevated tumor accumulation by targeted delivery. All these results suggest that Pc-GE11 can accumulate in EGFR-overexpressed tumor and it is a promising

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fluorescent probe for the detection of EGFR-rich tumor.

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Pc-GE11 Pc-rGE11

100 80 60 40 20 0 10

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30

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50

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Normalized Integrated Intensity 2 (Counts per mm )

(c)

Time (h)

Fig. 7. Fluorescence images of A431 tumor-bearing nude mice before and after intravenous

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injection of (a) Pc-GE11 and (b) Pc-rGE11 over 3 days. (c) Changes in fluorescence intensity per unit area of the tumor after treated with Pc-GE11 and Pc-rGE11. Data are expressed as

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mean value ± standard deviation (S.D.) of five mice.

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Pc-GE11 Pc-rGE11

30 25

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20

**

15 10 5

Tu m or

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Organs

Sk in

Ki dn ey (L ) Ki dn ey (R )

Lu ng

Sp le en

Li ve r

H ea rt

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Fluorescence Intensity (a.u.)

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Fig. 8. Ex vivo fluorescence intensity of different organs. The mice were sacrificed 3 days after the treatment of Pc-GE11 or Pc-rGE11. Data are expressed as mean value ± standard deviation (S.D.) of five mice. Statistical significance of the fluorescence intensity at the tumor region: P

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value **p < 0.01, compared between the Pc-GE11 group and Pc-rGE11 group is calculated through variance analysis by Microsoft excel.

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3. Conclusions

An EGFR-targeted zinc(II) phthalocyanine (Pc-GE11) has been synthesized and

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characterized. The photophysical properties, cellular uptake, in vitro cytotoxicity and in vivo biodistribution of Pc-GE11 have been examined. Competitive assay showed that pretreatment with free GE11 peptide could block the cellular uptake of the conjugate in A431 cells, indicating the receptor-mediated internalization pathway. In addition, MTT assay showed that this conjugate was non-toxic in dark while it induced cytotoxicity upon irradiation. Pc-GE11 also exhibited preferential tumor accumulation in tumor-bearing mice. In summary, Pc-GE11 is a promising EGFR-targeted PDT agent and fluorescent probe. 14

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4. Experimental section

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4.1 Materials All the reactions were performed under an atmosphere of nitrogen. DMF was pre-dried with barium oxide and distilled under reduced pressure. n-Pentanol was distilled from sodium under reduced pressure. Tetrahydrofuran (THF) was dried by sodium and distilled under atmospheric

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pressure. All other solvents and reagents were of reagent grade and used as received. Chromatographic purifications were performed on silica gel (Macherey-Nagel, 230-400 mesh)

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columns with specific eluents. Size exclusion chromatography was carried out on Bio-Rad BioBeads S-X1 beads (200−400 mesh) with THF as the eluent. The GE11 peptide resin [FmocTyr(tBu)-His(Trt)-Trp(Boc)-Tyr(tBu)-Gly-Tyr(tBu)-Thr(Trt)-Pro-Gln(Trt)-Asn(Trt)-Val-Ileresin] and the scrambled sequence peptide resin [Fmoc-Tyr(tBu)-Trp(Boc)-Gly-Pro-Asn(Trt)-

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Ile-His(Trt)-Tyr(tBu)-Tyr(tBu)-Thr(Trt)-Gln(Trt)-Val-resin] were prepared manually following SPPS protocol [58] with commercially available N-α-Fmoc-protected amino acids. Sieber amide

described [57].

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resin (Novabiochem®) was used as the solid support. Compound Pc-COOH was prepared as

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4.2 Characterization

Reversed-phase HPLC was performed on a XBridge C18 column (5 µm, 4.6 mm × 150 mm) for analytical purpose or a XBridge C18 column (5 µm, 10 mm × 250 mm) for preparative purpose using a Waters system equipped with a 1525 binary pump and a Waters 2998 diode array detector. Electrospray ionization (ESI) mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer. UV−Vis and steady-state fluorescence spectra were taken on a Cary

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5G UV−vis−NIR spectrophotometer and a Hitachi F-7000 spectrofluorometer, respectively. The 1

O2 generation in PBS was measured by the method of chemical quenching of 1,3-

diphenylisobenzofuran (DPBF) [59]. The light source for singlet oxygen generation came from a

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300 W halogen lamp after passing through a water tank for cooling and a color glass filter (Newport) cut-on 610 nm.

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4.3 Synthesis of Pc-GE11

The Fmoc protecting group of tyrosine was first removed from the GE11 peptide resin (7.2 µmol)

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with piperidine (20%) in DMF, then the peptide resin was washed with DMF (5 mL × 2) and CH2Cl2 (5 mL × 2). A mixture of Pc-COOH (22 mg, 21.4 µmol), HOBt hydrate (3 mg, 19.6 µmol), and HBTU (8 mg, 21.1 µmol) in DMF (3 mL) was added to the peptide resin in the presence of DIPEA (10 µL). The mixture was bubbled with nitrogen overnight at room

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temperature. The dark-green resin was washed with DMF and CH2Cl2 and then dried in vacuo. The resin was then treated with 1.5 mL of cocktail solution containing TFA (88%), phenol (5%), TIS (2%) and H2O (5%) to remove all the protecting groups on the peptide sequence and cleave

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the peptide from the resin. The crude Pc-GE11 conjugate was purified by semi-preparative HPLC. The conditions were set as follows: solvent A = acetonitrile; solvent B = 0.1% TFA in

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distilled water; gradient: maintained at 30% A + 70% B in the first 5 min, then changed to 100% A in 15 min and maintained at 100% A for 3 min, then changed back to 30% A + 70% B in 3 min and maintained for another 5 min. The flow rate was fixed at 1 mL min-1 for analysis and 3.5 mL min-1 for purification. The purity of the conjugate was found to be >97% as determined by HPLC. HRMS (ESI): m/z calcd for C125H145N29O27Zn [M + 2H]2+, 1275.0087; found, 1275.0091.

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4.4 Synthesis of Pc-rGE11 The synthesis and purification of Pc-rGE11 was performed similarly according to the above procedure for Pc-GE11. The purity of the conjugate was found to be >97% as determined by

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HPLC. HRMS (ESI): m/z calcd for C125H143N29O27ZnNa2 [M + 2Na]2+, 1296.9906; found, 1296.9910.

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4.5 Cell lines and culture conditions

The human epidermoid carcinoma A431 (ATCC® CRL-1555™) and human breast

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adenocarcinoma MCF7 (ATCC® HTB-22™) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher, cat. no. 11965092). The medium was supplemented with fetal bovine serum (10%) and penicillin-streptomycin (100 units mL-1 and 100 µg mL-1

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respectively). The cells were kept at 37 oC in a humidified 5% CO2 atmosphere.

4.6 Flow cytometric analysis

Approximately 5 x 105 A431 cells or MCF7 cells per well in 2 mL cell culture media were added

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in 35 mm Petri dish and incubated overnight. The cells were then treated with Pc-GE11 or PcrGE11 (4 µM) in growth medium (2 mL) for 4 h. The cells were rinsed with PBS (1 mL × 2) and

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harvested by trypsin (0.5 mL, 5 min). The activity of the trypsin was then quenched by culture medium (1 mL). The cells were collected by centrifugation at 1800 rpm for 3 min. The pellet was washed with PBS (1 mL) and then subject to centrifugation. The cells were suspended in 1 mL of ice cold PBS in dark and their fluorescence (λex = 638 nm, λem = 687-737 nm) was measured by flow cytometer (Beckman Coulter CytoFLEX S Flow cytometer analyzer) with 104 cells counted in each sample. For the competitive assays, A431 cells were pre-incubated with

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GE11 peptide (100 µM or 400 µM) for 2 h, then Pc-GE11 (4 µM) was added and incubated for another 4 h.

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4.7 Fluorescence microscopic studies

About 6 × 105 A431 cells per well in 2 mL cell culture media were added in 35 mm glass-bottom confocal dishes (MatTek) and incubated overnight. Stock solution of Pc-GE11 (2 mM) was

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prepared in DMSO and it was further diluted to 4 µM with culture medium. The cells were then treated with Pc-GE11 (4 µM, 2 mL) for 4 h. The cells were rinsed with PBS (1 mL × 2) and

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observed with a confocal laser scanning microscope (Zeiss Laser Scanning Microscope LSM 880 NLO with Airyscan) equipped with a 633 nm laser (Helium-Neon 633 laser). The emission signals at 638-747 nm were collected.

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4.8 MTT assay

Approximately 3 × 104 A431 cells per well in 100 µL cell culture media were added to 96-well plates and incubated overnight. Pc-GE11 was first dissolved in DMSO to give 2 mM solution,

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which was diluted to different concentrations with culture medium. The cells were then treated with different concentrations of Pc-GE11 in growth medium for 4 h. The cells were rinsed with

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PBS (100 µL × 2) and re-fed with 100 µL fresh media. The plate for dark cytotoxicity assay was directly plated in incubator overnight, while the other plate for photocytotoxicity testing was illuminated for 30 min at room temperature. The light source consisted of a 300 W halogen lamp, a water tank for cooling and a color glass filter (Newport, cut-on 610 nm). The fluence rate was 18 mW cm-2. An illumination of 30 min led to a total fluence of 32.4 J cm-2. After illumination, the cells were incubated overnight. Cell viability was determined by means of the colorimetric

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MTT assay.60 After rinsing the cells with PBS (100 µL), an 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution in PBS (3 mg ml-1, 50 µL) was added to each well followed by incubation for 4 h under the same conditions. DMSO (100 µL) was then added to

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each well to lyse the cells. The plates were placed on microplate reader (BioTek Synergy™ H1) at ambient temperature and the absorbance at 490 nm at each well was taken. The average absorbance of the blank wells, which did not contain the cells, was subtracted from the reading

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of the other wells. The cell viability was then determined by the equation: % Viability = [Σ(Ai/Acontrol × 100)]/n, where Ai is the absorbance of the ith data (i = 1, 2, …, n), Acontrol is the

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absorbance of the control wells, in which the drug was absent, and n is the number of data points.

4.9 In vivo study

Female Balb/c nude mice (20-25 g) were obtained from the Laboratory Animal Services Centre

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at The Chinese University of Hong Kong. All animal experiments had been approved by the Animal Experimentation Ethics Committee of the University. The mice were kept under pathogen-free conditions with free access to food and water. A431 cells (5 × 106 cells in 200 µL)

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suspended in Hank’s balanced salt solution (HBSS) were inoculated subcutaneously on the back of the mice and waited until the tumors had grown to a size of 60-100 mm3. Pc-GE11 or Pc-

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rGE11 (40 nmol) were first dissolved in DMSO (10 µL) and then diluted with distilled water (390 µL). These phthalocyanine solutions (200 µL, equivalent to 20 nmol) were intravenously injected into the tail vein of the tumor-bearing mice. In vivo fluorescence imaging was captured before and after the injection at different time points for 3 days with an Odyssey infrared imaging system (excitation wavelength at 680 nm, emission wavelength at ≥ 700 nm). The mice were sacrificed after 3 days and the organs were harvested. The ex vivo biodistribution of the

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compounds was determined using the fluorescence intensity obtained by the Odyssey infrared imaging system. The images were digitized and analyzed by the Odyssey imaging system

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software (no. 9201-500). Five mice were used for each compound.

Acknowledgements

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This work was supported by the City University of Hong Kong (ref. no. 7004585).

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