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

1

cRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for Tumor

2

Targeting in Photodynamic Therapy

3

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

6

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

9

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

10

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.

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

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

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

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[email protected] (Z. Hong).

16

‡

17

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

18

conjugates]. See DOI:

19

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

‡These authors contributed equally to this work.

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ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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

23

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

24

seriously restricts its clinical applications. A higher accumulation of photosensitizers

25

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

26

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

37

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

38

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

39

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

40

synthesized conjugate significantly improved the tumor enrichment and tumor

41

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

42

through one-time PDT treatment.


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43 44

Keywords: Photodynamic therapy, Pyropheophorbide a, Integrin, Peptide

45

conjugate, Photosensitizer

46 47

1. Introduction

48

Chlorophyll-a derivatives are very promising photosensitizers for photodynamic

49

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

50

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

51

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

52

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

53

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

54

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

55

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

57

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

58

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

59

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

60

tumor-accumulation ability; Npe67,

61

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

62

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

63

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

64

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

8

(talaporfin, also named as laserphyrin), a



65

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

66

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

67

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

68

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

69

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

70

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

73

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

75

solubility of properly selected highly hydrophilic peptide can markedly increase the

76

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

77

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

83

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

84

associated with Pyro could be solved with such conjugation.

85

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

86

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


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87

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

88

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

89

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

90

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

91

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

92

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

93

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

conjugate needs to be carefully tested and compared.

105

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

106

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

107

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

108

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



Page 6 of 44

109

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

110

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

111

treatment.

112 113 114

2. Materials and methods 2.1 Reagents

115

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

116

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

117

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

118

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

119

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

120

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

121

obtained

122

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

123

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

126

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

127

Fluorescence spectra were recorded using a Hitachi Model F-4500

128

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

129

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

130

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


FL

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131

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

132

system (IVIS Lumina II, Xenogen, USA).

133 134

2.3 Cell lines and animals

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

accordance with institutional guidelines of the Committee on Animals of Nankai

147

University (Tianjin, China).

148 149

2.4 Chemical syntheses

150

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

151

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

152

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



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153

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

154

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

155

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

156

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

157

to

158

dichloromethane and dried under vacuum.

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

temperature and was washed with DMF and dichloromethane. Cleavage and

170

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

171

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

172

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

173

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

174

purification.

afford

resin-cRGD-Linker-NH2,

after

being

washed


with

DMF

and

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175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

after HPLC purification.

185 186

2.5 Photophysical and photochemical properties

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

published procedure.36

195 196

2.6 In vitro photodynamic activities as determined by the MTT assay



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197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

later,

207

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

208

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

209

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

210

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

211

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

212

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

213

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

217

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

218

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


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219

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

221

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

225

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

226

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

227

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

229

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.

238

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

239

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



241

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

242

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

243

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

250

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

252

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

253

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

260

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.

262


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263

2.11 Statistical analysis

264

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

cRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for

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