cRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for

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cRGD Peptide-Conjugated Pyropheophor¬bide-a Photosensitizers for Tumor Targeting in Photodynamic Therapy Wenjing Li, Sihai Tan, Yutong Xing, Qian Liu, Shuang Li, Qingle Chen, Min Yu, Fengwei Wang, and Zhangyong Hong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01064 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Molecular Pharmaceutics

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cRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for Tumor

2

Targeting in Photodynamic Therapy

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Wenjing Li,‡a Sihai Tan,‡b Yutong Xing,a Qian Liu,a Shuang Li,a Qingle Chen,a Min Yu,c,* Fengwei Wang,d,* Zhangyong Hong a,**

4

5

a

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Protein Sciences, College of Life Sciences, Nankai University, Tianjin 300071, P. R.

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China. bTianjin University of Traditional Chinese Medicine, Tianjin 300193, P. R.

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China. cDepartment of Gynecologic Oncology, Tianjin Medical University Cancer

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Institute and Hospital, National Clinical Research Center for Cancer, Tianjin 300060,

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State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of

P. R. China. dPeople’s Hospital of Tianjin, Tianjin 300180, P. R. China.

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*Corresponding author. Tel.: +86 022 23340123-3111.

12

**Corresponding author. Tel.: +86 022 23498707.

13

***Corresponding author. Tel.: +86 022 23003610

14

E-mail addresses: [email protected] (M. Yu); [email protected] (F. Wang);

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[email protected] (Z. Hong).

16



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resin-cRGD-NH2 and cRGD peptide, HPLC chromatogram and ESI-HRMS of the

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conjugates]. See DOI:

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Electronic Supplementary Information (ESI) available: [Synthetic procedure for

‡These authors contributed equally to this work.

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Abstract

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Pyropheophorbide-a (Pyro) is a highly promising photosensitizer for tumor

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photodynamic therapy (PDT), although its very limited tumor-accumulation ability

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seriously restricts its clinical applications. A higher accumulation of photosensitizers

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is very important for the treatment of deeply seated and larger tumors. The

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conjugation of Pyro with tumor-homing peptide ligands could be a very useful

27

strategy to optimize the physical properties of Pyro. Herein, we reported our studies

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on the conjugation of Pyro with a cyclic cRGDfK (cRGD) peptide, an integrin

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binding sequence, to develop highly tumor-specific photosensitizers for PDT

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application. To further reduce the non-specific uptake and, thus, reduce the

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background distribution of the conjugates in normal tissues, we opted to add a highly

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hydrophilic polyethylene glycol (PEG) chain and an extra strongly hydrophilic

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carboxylic acid group as the linker to avoid the direct connection of the strongly

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hydrophobic Pyro macrocycle and cRGD ligand. We reported here the synthesis and

35

characterization of these conjugates, and the influence of the hydrophilic modification

36

on the biological function of the conjugates was carefully studied. The

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tumor-accumulation ability and photodynamic-induced cell-killing ability of these

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conjugates were evaluated through both in vitro cell-based experiment and in vivo

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distribution and tumor therapy experiments with tumor-bearing mice. Thus, the

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synthesized conjugate significantly improved the tumor enrichment and tumor

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selectivity of Pyro, as well as abolished the xenograft tumors in the murine model

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through one-time PDT treatment.

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Molecular Pharmaceutics

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Keywords: Photodynamic therapy, Pyropheophorbide a, Integrin, Peptide

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conjugate, Photosensitizer

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1. Introduction

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Chlorophyll-a derivatives are very promising photosensitizers for photodynamic

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therapy (PDT).1-4 Compared with the clinically used first-generation photosensitizer

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Photofrin® (Aptalis, Birmingham, AL, USA; approved in 1997; 1170 L mol-1 cm-1 of

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extinction coefficient at 630 nm),5, 6 these derivatives have obvious advantages to be

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used as photosensitizers. They always have a greater than 50% singlet oxygen

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quantum yield and a more than thirty-fold higher extinction coefficient of Photofrin at

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a longer absorption wavelength (>660 nm).7 Currently, several chlorophyll-a

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derivatives have been approved by the Food and Drug Administration (FDA) or are in

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various stages of development for clinical PDT use.8 For example, verteporfin9 (also

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known as Visudyne®, a product of Novartis pharmaceuticals) has been used clinically

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for ocular PDT of age-related macular degeneration (AMD) worldwide since 2000,

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although much of its intention in cancer treatment10 failed, likely due to relatively low

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tumor-accumulation ability; Npe67,

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hydrophilic chlorophyll-a derivative mainly for the presence of an aspartyl residue,

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has been approved in Japan (in 2004) for the treatment of early centrally located lung

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cancer; radachlorins9 (Rada-Pharma Ltd., Russia), an aqueous solution of three

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chlorins derived from chlorophyll-a, is in clinical trials for several cancer indications

8

(talaporfin, also named as laserphyrin), a

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in Russia; and HPPH10-12 (photochlor) is currently in advanced clinical trials.

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Compared with many other chlorophyll-a derivatives, pyropheophorbide-a13, 14 (Pyro)

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is one of the most interesting ones to be used as a photosensitive because of its

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relatively much higher stability and easier preparation. However, considering the

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druggability of Pyro, several problems presented seem to seriously restrict its clinical

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applications. Pyro has very limited tumor-accumulation ability and very limited water

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solubility, resulting in a relatively narrow therapeutic window and severe cutaneous

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photosensitivity due to skin accumulation.15

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The conjugation of Pyro with tumor-homing peptide ligands could be a very

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useful strategy to optimize the physical properties of Pyro.16-19 The high water

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solubility of properly selected highly hydrophilic peptide can markedly increase the

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water solubility of the whole conjugates. Additionally, the receptor-targeting

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capability of the tumor-homing peptide ligands can mediate the tumor-specific

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internalization of Pyro, thus increasing the tumor accumulation and selectivity of

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Pyro.15 A higher accumulation of photosensitizers can increase the response of the

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photosensitizers to the relatively subdued excitation light in deeper positions;20, 21 thus,

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it will be helpful for the treatment of deeply seated and larger tumors. Additionally,

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the increased tumor-tissue selectivity will also be helpful to reduce the damage to

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adjacent normal tissues.22 Both the problems of water solubility and tumor enrichment

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associated with Pyro could be solved with such conjugation.

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Herein, we studied the optimization of Pyro by conjugation with a cyclic

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cRGDfK (cRGD) peptide23-27 (Fig. 1) to solve its tumor enrichment problem and

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Molecular Pharmaceutics

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water solubility problem. cRGD peptide has a high binding affinity for integrin

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receptors, which are generally overexpressed in many types of tumor cells or tumor

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vessels, but has limited expression in normal cells.28-30 The conjugation of cRGD

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peptide with Pyro could be an ideal strategy to improve the tumor-accumulation

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ability and water solubility of Pyro. However, the strong hydrophobic property of the

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Pyro macrocycle in the conjugate may significantly influence the receptor affinity of

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the cRGD ligand, likely reducing the tumor selectivity of the conjugate for PDT

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therapy. Thus, we opted to add a highly hydrophilic polyethylene glycol (PEG) chain

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as the linker and a further hydrophilic carboxylic acid group to incorporation into the

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conjugate between the Pyro macrocycle and peptide ligand to avoid the direct

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connection of the strong hydrophobic Pyro macrocycle and cRGD ligand and to

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further increase the water solubility of the whole conjugate. The resulting high

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solubility characteristic of the conjugates could greatly reduce non-specific uptake as

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well as the background distribution of the conjugates in the normal tissues.31-35 While

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highly hydrophilic modification will also have its disadvantages, it may reduce the

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PDT activity of photosensitizers via reduction of the membrane attachment. Thus,

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how the incorporation of hydrophilic groups influences the biological function of the

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conjugate needs to be carefully tested and compared.

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We reported herein the synthesis and characterization of these conjugates. Their

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tumor-accumulation ability and photodynamic-induced cell-killing ability were

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evaluated through both in vitro cell-based experiment and in vivo distribution and

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tumor therapy experiments with tumor-bearing mice. As a result, the optimized

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conjugate significantly improved the tumor enrichment and selectivity of Pyro, as

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well as abolished the xenograft tumors in the murine model through one-time PDT

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treatment.

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2. Materials and methods 2.1 Reagents

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N-Ethyl-N'-(3-dimethylaminopropyl)

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diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA) and triisopropylsilane (Tis)

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were purchased from Heowns (Tianjin, China). N-Hydroxybenzotriazole (HOBt),

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2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylhexafluorophosphate

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N,N′-carbonyldiimidazole (CDI) were purchased from GL Biochem Ltd. (Shanghai,

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China). Fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) were

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obtained

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3-Diphenylisobenzofuran (DPBF) and N,N-dimethyl-Formamide (DMF), were

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obtained from J&K Chemical Ltd. (Beijing, China).

from

Gibco

(NY,

carbodiimide

USA).

Other

hydrochloride

chemical

(EDC·HCl),

(HBTU),

reagents,

and

including

124 125

2.2 Instruments

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The Cary 5000 spectrophotometer (Varian Co., USA) was used for UV-Vis spectra.

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Fluorescence spectra were recorded using a Hitachi Model F-4500

128

spectrophotometer (Tokyo, Japan). Mass data were obtained using a Varian 7.0 T

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FTMS. High-performance liquid chromatography (HPLC) (Shimadzu, Japan) was

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used for the purification and analysis of the conjugates. In vivo imaging analysis was

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FL

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performed at different time points after injection using an IVIS Lumina Imaging

132

system (IVIS Lumina II, Xenogen, USA).

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2.3 Cell lines and animals

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Human glioblastoma U87-MG cells and human prostate carcinoma PC3 cells were

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purchased from a typical culture preservation commission cell bank of the Chinese

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Academy of Sciences (Shanghai, China). Human epidermoid carcinoma A431 cells

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and mouse breast carcinoma 4T1 cells were obtained from the American Type Culture

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Collection. U87-MG cells, PC3 cells and 4T1 cells were grown in DMEM culture

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medium, and A431 cells were cultured in RPMI 1640 culture medium. All media

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contained L-glutamine and were supplemented with 10% fetal bovine serum (FBS)

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and 1% Pen/Strep (10,000 U penicillin, 10 mg streptomycin). A solution of 0.05%

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trypsin and 0.25% EDTA in PBS was used for cell detachment.

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BALB/c nude mice (male and female, 7–8 weeks old) were provided by Beijing Vital

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River Laboratory Animal Technology Co., Ltd. The studies were carried out in

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accordance with institutional guidelines of the Committee on Animals of Nankai

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University (Tianjin, China).

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2.4 Chemical syntheses

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Synthesis of resin-cRGD-Linker-NH2: The cRGD peptide on the resin with the

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arginine side chain Pbf protected and lysine side chain amine free (resin-cRGD-NH2)

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was prepared according to the procedure in the Supporting Information section.

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Resin-cRGD-NH2 (1.0 g, 0.5 mmol) was reacted with diglycolic anhydride (116.1 mg,

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1.0 mmol) in 5 mL dimethylformamide (DMF) for 5 hours to yield resin-cRGD-CO2H.

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Then, resin-cRGD-CO2H was activated twice with 0.5 M CDI for 1 hour, followed by

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agitating with 0.5 M HOBt and di-(3-aminopropyl) digol (1.1 g, 5 mmol) for 5 hours

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to

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dichloromethane and dried under vacuum.

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Synthesis of resin-cRGD-Linker-Glu-NH2: To resin-cRGD-Linker-NH2 (1.0 g, 0.5

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mmol) in 5 mL of DMF were added Fmoc-Glu(OtBu)-OH (0.32 g, 0.75 mmol),

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HBTU (0.38 g, 0.1 mmol), HOBt (0.1 g, 0.75 mmol) and DIPEA (0.26 g, 2 mmol).

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The mixture was agitated for 5 h and was washed with DMF and dichloromethane.

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The Fmoc group was then removed with 20% piperidine in DMF, followed by

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washing and drying under a vacuum, to produce resin-cRGD-Linker-Glu-NH2.

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Synthesis of RGD-L-Pyro 1: Resin-cRGD-Linker-NH2 (18.6 mg, 0.0093 mmol) was

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suspended in DMF (1.0 mL) for 15 minutes before adding a mixture of Pyro (10.0 mg,

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0.018 mmol), EDC (5.4 mg, 0.028 mmol), HOBt (5.1 mg, 0.037 mmol) and DIPEA

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(15.5 µL, 0.09 mmol) in 200 µL of DMF. The mixture was shaken overnight at room

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temperature and was washed with DMF and dichloromethane. Cleavage and

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de-protection were performed by treatment of the dried resin with 200 µL of cleavage

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solution (TFA/Tis/water = 95:2.5:2.5) for 40 minutes at room temperature. The

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product was precipitated and washed four times with ether (2.0 mL) and then was

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dried under reduced pressure to produce RGD-L-Pyro 1 (7.8 mg, 58.4%) after HPLC

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purification.

afford

resin-cRGD-Linker-NH2,

after

being

washed

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DMF

and

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Synthesis of RGD-L-Glu-Pyro 2: Resin-RGD-Linker-Glu-NH2 (18.6 mg, 0.0093

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mmol) was suspended in DMF (1.0 mL) for 15 minutes before adding a mixture of

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Pyro (10.0 mg, 0.018 mmol), EDC (5.4 mg, 0.028 mmol), HOBt (5.1 mg, 0.037 mmol)

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and DIPEA (15.5 µL, 0.09 mmol) in 200 µL of DMF. The mixture was shaken

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overnight at room temperature and was washed with DMF and dichloromethane.

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Cleavage and de-protection were performed by treatment of the dried resin with 200

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µL of cleavage solution (TFA/Tis/water = 95:2.5:2.5) for 40 minutes at room

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temperature. The product was precipitated and washed four times with ether (2.0 mL)

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and was dried under reduced pressure to produce RGD-L-Glu-Pyro 2 (8.3 mg, 57.0%)

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after HPLC purification.

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2.5 Photophysical and photochemical properties

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The spectroscopic properties of RGD-L-Pyro 1, RGD-L-Glu-Pyro 2 and free Pyro

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were measured according to our published procedures.36 Absorption spectra were

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recorded from 500 nm to 800 nm. Fluorescence excitation and emission spectra were

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recorded from 500 to 800 nm upon emission at 680 nm and excitation at 670 nm using

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a Hitachi Model F-4500 FL spectrophotometer (Tokyo, Japan). The singlet oxygen

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quantum yield (Φ∆) of RGD-L-Pyro 1 and RGD-L-Glu-Pyro 2 was measured using

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the singlet oxygen quencher DPBF in DMF with Pyro as a reference according to our

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published procedure.36

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2.6 In vitro photodynamic activities as determined by the MTT assay

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The in vitro PDT activities of the conjugates to αvβ3 over-expressing cancer cells were

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investigated using αvβ3-high expression cell lines (human glioblastoma U87-MG cells,

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human prostate carcinoma PC3 cells, and mouse breast carcinoma 4T1 cells), with

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human epidermoid carcinoma A431 cells that have low expression of αvβ3 receptors

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selected as a negative control. The cancer cells were seeded at a density of 5000 cells

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per well in a 96-well plate and were cultured for 24 h in a standard humidified cell

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culture incubator (37 °C, 5% CO2) prior to PDT. Next, the cells were incubated with

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different concentrations of the conjugates for 4 h, followed by illumination with a

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660-nm laser (40 mW/cm2, 10 min) or incubation in the dark as controls. Four hours

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later,

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(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium

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concentration of 0.5 mg/mL) was added, and then, the cells were cultured for another

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4 hours. Subsequently, the resulting methylenezine crystal was dissolved with 100

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microliters of dimethyl sulfoxide (DMSO), and the absorbance was measured at 490

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nm. The dark toxicity test was the same as that of the above operation, except that it

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was not illuminated. EC50 (concentration at which cell viability was decreased by

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50%) values were calculated using GraphPad Prism software (GraphPad Software

214

Inc., San Diego, CA, USA).

100

µL

of

fresh

culture

medium

supplemented

with

bromide,

MTT final

215 216

2.7 Competitive inhibition assay

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Approximately 1× 104 PC3 cells per well were plated in a 96-well plate for 24 hours.

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The cells were pre-incubated with 1.0 mM unmodified cRGD peptide for 2 hours.

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Next, different concentrations of conjugates or free Pyro were added in the 96-well

220

plate. Four hours later, the plates were irradiated with 660-nm light (40 mW/cm2, 10

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min). After 24 h, MTT was added. The subsequent operation was the same as that

222

described in the in vitro photodynamic activity assay.

223 224

2.8 Confocal microscopy

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Confocal microscopy was used to monitor the cellular binding and distribution of the

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conjugates RGD-L-Pyro 1, RGD-L-Glu-Pyro 2 and free Pyro. PC3 cells were seeded

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in a 24-well plate paved with sterile coverslips at a density of 5×104 cells/well. After

228

overnight culture, the conjugates were added to a final concentration of 1.0 µM in a

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regular growth medium, and the cells were incubated under standard conditions for an

230

additional 4 hours. For the competition experiments, the cells were preincubated at

231

37 °C for 2 h with a 1000-fold excess (1.0 mM) of unmodified cRGD peptide before

232

addition of the conjugates. After paraformaldehyde fixation, nuclei were stained with

233

DAPI (blue fluorescence). Then the cells were washed three times with PBS buffer

234

and observed using a Leica TCS SP8 Confocal Microscope (Leica, Wetzlar,

235

Germany).

236 237

2.9 In vivo NIR optical imaging.

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Male or female BALB/c nude mice (7-8 weeks of age) received subcutaneous

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implants consisting of 4×106 U87-MG cells, PC3 cells or A431 cells in their right

240

back. When the tumor volumes reached up to 100 mm3, the mice were divided into

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three groups, with each group injected with the conjugates RGD-L-Pyro 1,

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RGD-L-Glu-Pyro 2 or free Pyro at a dose of 30 nmol per mouse via the tail vein. In

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vivo fluorescence analysis was performed before and after the injection at various

244

time points using the IVIS Lumina Imaging system (IVIS Lumina II, Xenogen, USA)

245

with a Cy5.5 filter (excitation: 615–665 nm, emission: 695–770 nm). If the organs

246

were imaged, the mice were euthanized at 24 h post-injection, and the tumor grafts

247

were collected for the acquisition of fluorescence images.

248 249

2.10 In vivo antitumor activity

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The antitumor effect was verified by xenografts in nude mice. Each nude mouse was

251

subcutaneously inoculated with 4×106 PC3 cells in the right back. When the tumors

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reached an average of approximately 100 mm3, the mice were randomly divided into

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two groups (4 mice per group). The experimental group was administered

254

intravenously with RGD-L-Glu-Pyro 2 (30 nmol per mouse), followed by irradiation

255

(200 mW/cm2, 670 nm, 15 min) 3 hours after the injection. The control group was

256

injected with PBS similarly, followed by the same irradiation 3 hours after the

257

injection. The animals were monitored for the tumor volume and body weight for 60

258

days sequentially or until the tumor size reached 1000 mm3. The tumor size was

259

monitored every two days using a digital caliper, and tumor volumes were calculated

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according to the formula: tumor volume (mm3) = length×(width)2/2. To observe the

261

treatment of the mice visually, the mice were imaged every two days.

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2.11 Statistical analysis

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All data are expressed as the means ± standard error of the mean (SEM), and the

265

differences were determined by Student’s t-test. P