Targeting Phosphatidylserine with a 64Cu-Labeled Peptide for

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Targeting phosphatidylserine with a 64Culabeled peptide for molecular imaging of apoptosis Amanda Perreault, Susan Richter, Cody Nils Bergman, Melinda Wuest, and Frank Wuest Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00666 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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Molecular Pharmaceutics

Targeting phosphatidylserine with a 64Cu-labeled peptide for molecular imaging of apoptosis -Article-

Amanda Perreault, Susan Richter, Cody Bergman, Melinda Wuest, Frank Wuest*

Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, AB T6G 2X4, Canada

Keywords:

64

Cu, phosphatidylserine, peptide, apoptosis, positron emission tomography

(PET)

*Corresponding Author: Frank Wuest ([email protected]) 1

ACS Paragon Plus Environment


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ABSTRACT: Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PSbinding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 (64Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric Cu to prepare radiotracer

64

NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6 and

nat

acid (Ava) linker for subsequent radiolabeling with

64

Cu-

Cu-

NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer

64

Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic

EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide

64

Cu-NOTA-Ava-

PSBP-6 was synthesized in high radiochemical yields of >95%. The IC50 values for PSbinding potency of PSBP-6, NOTA-Ava-PSBP-6 and

nat

Cu-NOTA-PSBP-6 were 600

µM, 30 µM and 23 µM, respectively. A competitive radiometric cell binding assay confirmed binding of

64

Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4

2+

cells in a Ca -independent manner. PET imaging studies demonstrated significantly higher uptake of

64

Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV5min 0.95 ±

0.04) compared to control tumors (SUV5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-AvaPSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer 64Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional “fine-tuning” of

64

Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding

potency and in vivo stability to improve tumor uptake and retention. 2


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INTRODUCTION Dysregulation of cell death plays a prominent role in tumor development and cancer progression.1 The desired effect of anticancer therapy is to induce tumor cell death, and there are several different cell death pathways that may be induced.2 Molecular imaging agents that can non-invasively detect a type of cell death in vivo would provide valuable information on tumor viability and response to therapy. However, there are currently no clinically approved, widely used molecular imaging agents that target biomarkers of cell death pathways. The development of cell death imaging agents is an important and intensively explored area of current preclinical and clinical research. Extensive studies of the molecular mechanisms underlying cell death led to the identification of various

biomarkers for molecular targeting of cell death through

molecular imaging probes. Processes and biomarkers of cell death include DNA fragmentation, caspase 3/7 activation, mitochondrial membrane depolarization, plasma membrane depolarization, and loss of plasma membrane integrity.3 Phosphatidylserine (PS), a membrane phospholipid normally confined to the inner leaflet of the cell membrane, becomes externalized to the outer leaflet in cells undergoing apoptosis, and sometimes in cells undergoing other modes of cell death.4 Since PS is present on the outside of cells undergoing cell death, this phospholipid is an attractive molecular target for cell death-detecting probes.5 The most commonly known PS-targeting agent is the endogenous, 36 kDa protein annexin V, which tightly binds to PS with nanomolar affinity in the presence of millimolar concentrations of calcium ions (Ca2+).6-8 Fluorescentlylabeled annexin V is commonly used in standard staining techniques to determine apoptosis levels in vitro.9 Extensive research was devoted to the development of a clinically suitable form of radiolabeled annexin V that can image cell death in vivo, with technetium-99m (99mTc)-labeled annexin V receiving the most attention. However, there are several challenges associated with the use of radiolabeled annexin V for in vivo imaging of cell death, which have limited its use in the clinic. These challenges include elaborate and complex radiolabeling procedures, poor biodistribution profiles displaying high non-specific uptake into the liver and kidneys, slow blood clearance resulting in poor image contrast, inability to control Ca2+ concentrations in vivo, poor tumor tissue

3



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penetration, and lack of knowledge of the optimal time for imaging after cell death has been induced and radiolabeled annexin V has been administered.3,10,11 PS-binding peptides, on the other hand, offer many advantages over PS-binding proteins such as annexin V. Due to their small size, peptides can penetrate tissues easily and are rapidly cleared from the blood, allowing for good image contrast. They have low immunogenicity and are relatively simple and inexpensive to synthesize.12 Since peptides can withstand harsher reaction conditions, procedures for modification and radiolabeling of peptides are usually more simple.13 In addition, peptides can be structurally modified to improve metabolic stability along with other pharmacokinetic and pharmacodynamic properties.14 Using phage display technology and other highthroughput peptide screening techniques, libraries of biologically active peptides can be produced.15 Using these technologies, several PS-binding peptides have been identified.16-23 Many of these PS-targeting peptides have shown promise when evaluated in preclinical tumor models of treatment-induced cell death.16,21,23-27 14-mer PS-binding peptide 6 (FNFRLKAGAKIRFG, PSBP-6), reported by Xiong et al., is a modified version of the synthetic 14-mer peptide PSBP-0 (sequence FNFRLKAGQKIRFG) originally identified and described by Igarashi et al. in 1995.22,28 PSBP-0 was derived from a conserved PS-specific binding site found on protein kinase C and PS decarboxylase, an enzyme that converts PS to phosphatidylethanolamine (PE).28 PSBP-0 was found to bind specifically to PS, but with low affinity. Xiong et al. later produced a library of 14-mer peptides based on the PSBP-0 sequence in order to identify a peptide with higher affinity for PS.22 A surface-plasmon resonance (SPR) biosensory assay was used to identify 14-mer peptide PSBP-6, which displayed high affinity for PS (Kd ~100 nM).22 Xiong et al. conjugated PSBP-6 with a single amino acid chelator (SAAC) on its Nterminal end, which was then labeled with

99m

tomography (SPECT) imaging of cell death. 99m

Tc for single photon emission computed

22

The ability of SPECT imaging agent

Tc-SAAC-PSBP-6 to target chemotherapy-induced tumor apoptosis was evaluated in

nude mice bearing B16/F10 murine melanoma tumors, using paclitaxel chemotherapy. In this study, biodistribution and autoradiography analyses revealed higher uptake of 99m

Tc-SAAC-PSBP-6 in paclitaxel-treated tumors compared to untreated tumors.22 4


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The same research group also conducted preclinical SPECT imaging studies in nude mice bearing B16/F10 melanoma tumors treated with paclitaxel, as well as in C3H/HeJ mice bearing 38C13 lymphoma tumors treated with cyclophosphamide. In both models of chemotherapy-induced tumor apoptosis, an increase in uptake of

99m

Tc-SAAC-PSBP-

6 was observed in treated tumors versus untreated tumors, further demonstrating the utility of radiopeptide

99m

Tc-SAAC-PSBP-6 for SPECT imaging of chemotherapy-

induced tumor cell death.29 Uptake of

99m

Tc-SAAC-PSBP-6 also negatively correlated

with [18F]FDG uptake in both tumor models, with [18F]FDG uptake significantly decreasing after treatment with chemotherapy.29 With the advantages that positron emission tomography (PET) technology offers over SPECT, such as higher sensitivity and better quality images, we envisaged to label reported high affinity PS-binding peptide PSBP-6 with a positron emitting radionuclide for PET imaging of cell death. In the present study, we describe the synthesis and radiopharmacological evaluation of PSBP-6 labeled with positron emitter copper-64 (64Cu). For this purpose, 14-mer peptide PSBP-6 was modified with an aminovaleric acid (Ava) linker and macrocyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). NOTA-Ava-PSBP-6 was radiolabeled with 64Cu to give radiotracer 64Cu-NOTAAva-PSBP-6. We evaluated PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6 and nat

Cu-NOTA-Ava-PSBP-6 in a competitive radiometric PS-binding assay using

labeled

annexin

V

as

radiotracer

Radiopharmacological evaluation of

and

annexin

V

as

internal

64

Cu-

reference.

64

Cu-NOTA-Ava-PSBP-6 involved studies on

binding to PS using apoptotic murine EL4 lymphoma cells, dynamic PET imaging experiments in EL4 tumor-bearing mice and metabolic stability studies in vivo. We also conjugated fluorescent molecule fluorescein isothiocyanate (FITC) to PSBP-6 and examined binding of FITC-conjugated PSBP-6 to EL4 cells undergoing camptothecininduced cell death using flow cytometry and fluorescence confocal microscopy.

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MATERIALS AND METHODS General. All chemicals and reagents were obtained from Sigma-Aldrich® unless otherwise stated. Purified recombinant wild-type human annexin V (1 mg or 5 mg, lyophilized) was purchased from BioVision (Milpitas, USA). 64

Cu was produced on a CS-15 biomedical cyclotron at Washington University School of

Medicine (St. Louis, MO) in the form of [64Cu]CuCl2 in 0.01 M HCl, which was usually diluted with 100 mM ammonium acetate (NH4OAc) buffer (pH 5.5). Water and buffers used for metal and radiometal chemistry procedures were first treated with Chelex® 100 resin (Bio-Rad) to eliminate heavy metal contamination. Radio-thin-layer chromatography (radio-TLC) detection was performed using a Bioscan AR-2000 Imaging Scanner. Semi-preparative high performance liquid chromatography (HPLC) was performed on a Gilson system consisting of a 321 pump and a 171 diode array detector, and a Berthold Technologies Herm LB 500 was used as radiodetector. Analytical HPLC was performed on a Shimadzu system equipped with a DGU-20A5 degasser, SIL-20A HT autosampler, LC-20AT pump, SPD-M20A photodiode-array detector, and Ramona Raytest radiodetector. Radiopeptides were subjected to analytical HPLC using a Phenomenex Luna® 10u C18(2) 100Å, 250 × 4.6 mm column and the following gradient of water/0.2% (v/v) trifluoroacetic acid (TFA) (A) and CH3CN (B): 0 min: 10% B, 10 min: 30% B, 10-17 min: 50% B, 17-23 min: 70% B, 23-27 min: 90% B, 27-30 min: 90% B (flow rate = 1 mL/min). UV absorbance was monitored at 210 nm and/or 254 nm. De-ionized water was obtained from a Barnstead Nanopure water filtration system (Barnstead Diamond Nanopure pack organic free RO/DIS). Quantification of radioactive samples during radiochemistry was achieved using a Biodex ATOMLAB™ 400 dose calibrator. Centrifugation of samples was done in a Hettich Zentrifugen Rotina 35R or a Fisher Scientific Mini Centrifuge. Radioactive samples were measured using a PerklinElmer 2480 Automatic Gamma Counter WIZARD2®. Cells stained with Tryptan Blue were counted using a Bio-Rad TC10™/ TC20™ Automated Cell Counter. Flow cytometry was carried out using a Fluorescence Activated Cell Sorter (BD FACSCaliber™) bench top analyzer. For fluorescence confocal microscopy, a Zeiss LSM 710 confocal microscope was used. 6


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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on IDGel™ Express IR121S-20 precast gels (12% tris buffer), which were run in a BioRad Mini PROTEAN® Tetra Cell with running buffer. Gels were run with a constant current of 30 mA for approximately 1 h, and Radio-SDS-PAGE gels were scanned on a Fujifilm BAS 5000 phosphor imager and analyzed by AIDA Image Analyzer Software Version 4.50. The gels were then stained with Coomassie® Brilliant Blue R-250 (BioRad) for 30 min at 37 °C, and left in destaining solution overnight. Bicinchoninic acid (BCA) protein assays were performed as instructed from the BCA Protein Assay Kit (Pierce™): standards containing bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (pH 7.4) were prepared at concentrations of 0, 50, 100, 200, 300, 400, 600, and 800 µg/mL. 25 µL of each concentration of standard was pipetted into a 96-well plate, as well as 25 µL of the sample with unknown protein content. A solution of 2% BCA Reagent B (Pierce™) in BCA Reagent A (Pierce™) was made, and 200 µL of this solution was added to each well containing standard or sample. The plate was left to incubate for 25 min at 37 °C (Fisher Scientific Isotemp Incubator Model 546). Protein content in each well was then determined from absorbance measurements using a Molecular Devices Spectramax 340PC microplate reader. In-house preparations of buffers were as follows: Phosphate-buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 5.4 mM Na2HPO4, 570 µM KH2PO4, pH 7.4), 2.5 mM Ca2+ binding buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2), 1.25 mM Ca2+ binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, 1.25 mM CaCl2), Ca2+-negative binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl), running buffer for SDS-PAGE (25 mM Tris, 192 mM glycine, 0.1% SDS), Destaining solution (10% glacial acetic acid, 20% methanol, 70% deionized water).

7



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Peptide synthesis. All peptides were synthesized by Fmoc-based solid-phase peptide synthesis (SPPS) using a fully automated peptide synthesizer (Syro I, Multisyntech/Biotage). Rink Amide 4-methylbenzhydrylamine (MBHA) resin (100-200 mesh) was used as the solid support for synthesis of peptides FNFRLKAGAKIRFG-CONH2 (PSBP6),

NOTA-Bn-thiourea-Ava-FNFRLKAGAKIRFG-CONH2

(NOTA-Ava-PSBP-6),

and

FITC-Ava-PSBP-6. Pre-loaded resin (Rink Amide MBHA) was allowed to swell in 2 mL of dimethylformamide (DMF) for 15 min. Fluorenylmethyloxycarbonyl (fmoc) group deprotection was achieved by treatment with 40% piperidine/DMF for 5 min, followed by treatment with 20% piperidine/DMF for 15 min. Fmoc-protected amino acids (5 eq) were activated and coupled using 5 eq of O-benzotriazole-N,N,N’,N’-tetramethyl-uroniumhexafluoro-phosphate (HBTU), 5 eq of ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma), and 10 eq of N,N-diisopropylethylamine (DIPEA) over a 60 min time period followed by washing steps with DMF. Treatment with an acidic solution containing 87.5% TFA, 5% water, 5% thioanisole, and 2.5% 1,2-ethanedithiol (EDT) for 3.5 h induced cleavage of the assembled peptides from the resin with simultaneous deprotection of amino acid side chains. Resin was removed from the peptide solution through a syringe filter, and peptides were precipitated by the addition of ice-cold diethyl ether. Residual ether was removed by syringe filter, and the precipitated crude peptides were dried under vacuum. HPLC purification and subsequent lyophilization gave sufficiently pure peptides (>95% purity based on HPLC analysis) as white solids.

Synthesis of PSBP-6. PSBP-6 was synthesized according to the general procedure starting with 50 mg of Rink Amide MBHA resin. Peptide was purified by semipreparative HPLC using a gradient of water/0.2% TFA (A) and CH3CN (B), 2 mL/min: 05 min: 0% B, 10 min: 30% B, 25 min: 50% B, 30-40 min: 80% B. Yield: 32.6 mg (51%). Analysis: tR = 22.1 min. MW C77H122N24O15 calculated 1622.95, found LR-MS (ESI, positive) m/z 1623.9 [M + H]+.

8


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Synthesis of NOTA-Bn-thiourea-Ava-PSBP-6 (NOTA-Ava-PSBP-6). PSBP-6 was conjugated with metal chelating group NOTA via an aminovaleric acid (Ava) spacer. Ava-PSBP-6 was synthesized according to the general procedure using 50 mg of starting Rink Amide MBHA resin, with Ava as the N-terminal amino acid. Before the resin was cleaved, 22 mg of p-SCN-Bn-NOTA (1.1 equiv. to resin loading) and 55 µL (10 equiv. to resin loading) of triethylamine were added to the swelled resin in 0.5 mL of DMF. The resin mixture was left to react for 17.5 h at room temperature, after which the resin was washed and cleaved according to the general procedure. NOTA-Ava-PSBP-6 was purified by semi-preparative HPLC using the same gradient used to purify PSBP-6. Yield: 19 mg (22%). Analysis: tR = 26.9 min. MW C102H157N29O22S calculated 2172.2, found LR-MS (matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), positive) m/z 2173.4 [M + H]+.

Synthesis of

nat

Cu-NOTA-Ava-PSBP-6. Reference compound

nat

Cu-NOTA-Ava-PSBP-6

was synthesized by first adding 30 equiv. of CuCl2 to an excess of EDTA in 100 mM NH4OAc buffer (pH 6), which was shaken at room temperature for 30 min. 1 equiv. of NOTA-Ava-PSBP-6 (2 mg) in 100 mM NH4OAc buffer (pH 6) was then added to the CuCl2-EDTA mixture, and the reaction mixture was reacted at 60 °C for 1 h. The reaction mixture was then left to incubate at 37 °C overnight.

nat

Cu-NOTA-Ava-PSBP-6

was purified by semi-preparative HPLC using a gradient of water/0.2% TFA (A) and CH3CN (B), 2 mL/min: 0-10 min: 10% B, 10-25 min: 50% B, 25-30 min: 80% B, 30-40 min: 80% B. Analysis: tR = 26.8 min. The HPLC product was lyophilized and pure peptide was obtained as a light green powder. Yield 1.5 mg (73%). MW C102H154CuN29O22S calculated 2234.1, found LR-MS (MALDI-TOF, positive) m/z 2235.4 [M + H]+.

Synthesis of FITC-Ava-PSBP-6. The fluorescent compound FITC-Ava-PSBP-6 was prepared according to the general procedure. PSBP-6 was conjugated with FITC via an Ava spacer. 1 equiv. of FITC-NCS was added to the resin loading in 500 µL of 12:7:5 Pyridine:DMF:DCM, and the mixture was left to react overnight with protection from light. 9



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HPLC purification of FITC-Ava-PSBP-6 was accomplished using a gradient of water/0.2% TFA (A) and CH3CN (B), 2 mL/min: 0-5 min: 10% B, 10 min: 30% B, 25 min: 50% B, 30-40 min: 80% B. Yield: 13.9 mg (16.7%). Analysis: tR = 27.4 min. MW C103H142N26O21S calculated 2112.5, found LR-MS (MALDI-TOF, positive) m/z 2114.4 [M + H]+.

Radiochemistry Synthesis of

64

Cu-NOTA-annexin V.

64

Cu-NOTA-annexin V was synthesized according

to the procedure recently described by Wuest et al..27

Synthesis of

64

Cu-NOTA-Ava-PSBP-6. 10-32 µL aliquots containing 2 µg (1 nmol) of

NOTA-Ava-PSBP-6 were prepared. In order to label NOTA-Ava-PSBP-6 with

64

Cu, 100

µL of 100 mM NH4OAc buffer (pH 6) was first added to the aliquot, followed by 5-50 MBq of [64Cu]CuCl2 in 100 mM NH4OAc buffer (pH 6). The reaction mixture was incubated at 60 °C for 60 min. Quality control of the product was performed by semipreparative radio-HPLC using a Phenomenex LUNA® C18(2) (100Å, 250 × 10 mm, 10 µm) column and the following gradient of water/0.2% TFA (A) and CH3CN (B): 0-10 min: 10% B, 10-25 min: increase to 50% B, 25-30 min: increase to 90% B, 30-35 min: isocratic at 90% B (flow rate = 2 mL/min) (tR = 29.7 min), or analytical radio-HPLC using the analytical peptide gradient (tR = 16.4 min). Radio-TLC was performed on silica plates using 10 mM EDTA as solvent to give

64

Cu-NOTA-Ava-PSBP-6 in >95% radiochemical

purity (Rf = 0.0). No purification step was necessary, as radiochemical yields of >95% (RCP >95%) were obtained without purification, verified by all three quality control methods. The crude product in NH4OAc buffer (pH 5.5-6) was directly used for cell binding or PET imaging studies.

10


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Determination of lipophilicity. To determine the lipophilicity of radiopeptides, the shakeflask method was used.32 The partition coefficients of the radiopeptides were determined using n-octanol as organic phase and PBS (pH 7.4) as aqueous phase. 500 µL of each phase were added to a LoBind eppendorf tube, to which >1 MBq of radiopeptide was added, and the mixture was shaken vigorously for 5 min. The mixture was then centrifuged at 2000 rpm for 2 min to allow the layers to separate. Aliquots of 100 µL were removed from each phase and the amount of radiopeptide present in each phase was measured by a Wizard gamma counter. Experiments were performed in triplicate, and logD7.4 values were calculated. Competitive radiometric PS-binding assay. In order to examine the PS-binding potency of PSBP-6, as well as any changes in PS-binding potency induced by conjugation/labeling of the peptide, we performed a previously described competitive radiometric binding assay.27 0.3-0.5 µg of PS (1,2-dipalmitoyl-sn-glycero-3-phospho-Lserine sodium salt, Sigma-Aldrich) dissolved in 9:1 chloroform:methanol (further diluted in ethanol) was immobilized in each well of a BRANDplates® 96 well Strip Plate (Life Sciences). The plate was left overnight to allow the solvent to evaporate, forming a light film. To begin the assay, the wells were first washed twice with PBS to remove unbound PS. 5% non-fat dry milk (Bio-Rad) in PBS was used as blocking buffer. After 2 h incubation at room temperature, the blocking buffer was removed and wells were washed twice with PBS. 2.5 mM Ca2+ binding buffer was used to prepare serial dilutions of each peptide (10-7 to 10-3 M) as well as unmodified, wild-type annexin V (10-11 to 10-6 M). The unlabeled, wildtype annexin V was used as an internal reference, while used as competitive tracer.

64

Cu-NOTA-annexin V was

64

Cu-NOTA-annexin V was synthesized according to a

previously described procedure.27 200 µL of each concentration of PS-binding peptide or unmodified, annexin V was added to the wells in triplicate. 64Cu-NOTA-annexin V was diluted to an activity concentration of 1 kBq/µL, corresponding to 4.3 ng/µL of NOTAannexin V. 10 µL (10 kBq) of

64

Cu-NOTA-annexin V in PBS was added to each well,

incubating at room temperature for 2 h.

11



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The wells were then washed twice with Ca2+ buffer to remove unbound ligand, and the plate was separated into individual wells. Each well was placed into a scintillation vial, and bound activity was measured by a Wizard gamma counter. Maximum binding of 64Cu-NOTA-annexin V was determined by adding the radiotracer to wells that contained just PS and Ca2+ buffer, and no competing ligand. Nonspecific binding of

64

Cu-NOTA-annexin V was measured by adding the radiotracer to wells that

contained just Ca2+ buffer, with no immobilized PS. Data were analyzed as percent inhibition of

64

Cu-NOTA-annexin V binding.

Concentrations for half-maximum inhibition (IC50) were calculated from dose-response curves generated with GraphPad Prism® 5.0 software.

Cell uptake experiments. EL4 murine T-cell lymphoma cells (TIB-39™), which readily undergo apoptosis when introduced to cytotoxic agents, were purchased from the American Type Tissue Culture Center (ATCC) (Manassas, VA, USA) and maintained in RPMI 1640 medium (Gibco®) containing 20 mM HEPES, 10% fetal bovine serum (FBS) (Gibco®), 2 mM L-glutamine (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Cell density was maintained in the range of 2 x 105 cells/mL to 2 x 106 cells/mL, and cell growth medium was added or changed every other day. Cells were incubated at 37 °C in a humidified incubator with a 5% (v/v) CO2 atmosphere (ThermoForma Series II Water Jacketed CO2 Incubator). Induction of apoptosis in EL4 cells. In order to evaluate the ability of the radiopeptide to bind to apoptotic cancer cells, an in vitro model of chemotherapy-induced apoptosis was used, which was previously used to evaluate

18

F-labeled wild-type annexin V binding to

apoptotic cells.30 (s)-(+)-Camptothecin (Sigma-Aldrich), a DNA-topoisomerase I inhibitor, was used to induce apoptosis in EL4 cells. The optimal concentration of camptothecin used to induce apoptosis in EL4 cells over 24 h was determined by flow cytometry, using FITC-annexin V as an apoptosis marker.30 This in vitro model of cell death was used for the cell binding assays, flow cytometry experiments using FITCPSBP-6, and fluorescence confocal microscopy imaging. 12


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Cell binding assay. 22-24 h prior to the cell binding assay, two T75 flasks were prepared containing 15 mL of EL4 cells at a cell density of 1 million cells/mL. To one flask, 9 µL of the stock solution of camptothecin (2.5 mM in 1 M NaOH) was added to the 15 mL of medium to produce a final camptothecin concentration of 1.5 µM. To the control flask, 9 µL of 1 M NaOH solution was added. To prevent direct contact of 1 M NaOH and 2.5 mM camptothecin with the cells, NaOH/camptothecin was first added to the medium, which was then added to the cells. Control and treated cells were then left to incubate for one day at 37°C. On the day of experiment, control and treated cells were spun down, growth medium was removed, and cells were washed once with PBS, and once with binding buffer. In order to ensure that radiopeptide binding to PS was Ca2+-independent, two sets of binding assays were conducted: one set of binding assays were perfomed in Ca2+-negative binding buffer, and another set were performed in Ca2+-positive (2.5 mM CaCl2) binding buffer. Cells were resuspended in their respective binding buffer to 5 x 106 cells/mL. 300 µL of the cell suspensions were aliquoted into LoBind eppendorf tubes. 200 µL (~0.1 MBq) of radiopeptide diluted in the desired binding buffer was added to each eppendorf, incubating for 1, 15, 30 and 60 min. Binding was terminated at each time point by centrifuging the cells at 1500 rpm for 2 min. Most of the supernatant was removed by aspiration (~50 µL left behind), the tubes were spun down again (1500 rpm for 2 min), and the rest of the supernatant was removed with a pipette. The amount of radiopeptide bound to the cell pellets was measured by a Wizard gamma counter. To determine protein content, cells (control and treated) were lysed with CelLytic™ (200 µL) for 10 min at 4 °C, centrifuged at 4 °C at 10000 rpm for 8 min, and left in the freezer for at least one night. The lysate was then thawed and protein content was analyzed using the BCA protein assay.

Flow cytometry and confocal microscopy experiments. In order to determine the optimal concentration of camptothecin to induce apoptosis in EL4 cells, apoptosis levels in EL4 cells treated with increasing doses of the drug were measured using flow cytometry. The FITC Annexin V Apoptosis Detection Kit 1 (BD Pharmingen™) was used to detect apoptotic cells. 13



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From this kit, fluorescein isothiocyanate conjugated annexin V (FITC-annexin V), was used to stain apoptotic cells, while propidium iodide (PI) identified cells that had lost their membrane integrity (necrotic/late apoptotic cells). A 6-well plate was prepared with 3 mL of RPMI medium containing 3 million cells (cell density of 1 million cells/mL) in each well. Each well had a different concentration of camptothecin: 0.5 µM, 1.0 µM, 1.5 µM, and 2.0 µM, and the control well contained just NaOH without camptothecin. The cells were left to incubate for 22-24 h at 37 °C. The next day, the cells were centrifuged, washed twice with PBS, and resuspended in Ca2+-containing binding buffer (BD Pharmingen™) to a cell density of 1 million cells/mL. 100 µL of each cell solution was transferred to its own flow cytometry tube. To each tube, 3 µL of FITC-annexin V and 3 µL of PI were added. The tubes were gently shaken and left to incubate in the dark for 15 min at room temperature. 400 µL of binding buffer was then added to each tube, and apoptotic and necrotic cells in each tube were counted by a fluorescence activated cell sorter.

Flow cytometry using FITC-Ava-PSBP-6. Flow cytometry was also used to examine the ability of the fluorescent peptide FITC-Ava-PSBP-6 to bind to apoptotic cells. EL4 cells were treated with 1.5 µM camptothecin for 22-24 h. A fresh stock solution of 50-100 µM of FITC-PSBP-6 was prepared on the day of the experiment by dissolving the lyophilized peptide in DMSO and then diluting it in deionized water to achieve 95%) after HPLC purification. For complexation with Cu2+, peptide PSPB-6 was functionalized with macrocyclic chelator NOTA via an aminovaleric acid (Ava) spacer to give NOTA-Ava-PSPB-6 in 22% yield. Treatment of NOTA-Ava-PSPB-6 with CuCl2 afforded reference compound

nat

Cu-NOTA-Ava-PSBP-6 in 73% yield.

Structures of peptides PSBP-6, NOTA-Ava-PSBP-6 and

nat

Cu-NOTA-Ava-PSBP-6 are

given in Scheme 1.

((Scheme 1))

Binding potency of all three peptides to phospholipid PS was measured in a competitive radiometric binding assay recently developed in our research group.27 In the assay, binding of radiotracer

64

Cu-NOTA-annexin V to immobilized PS was challenged with

different concentrations of the peptides (PSBP-6, NOTA-Ava-PSBP-6 and

nat

Cu-NOTA-

Ava-PSBP-6), as well as with different concentrations of wild-type annexin V as internal reference compound with high affinity binding to PS. The results of the competitive radiometric PS binding assay are summarized in Figure 1.

((Figure 1))

Wild-type annexin V was found to have an IC50 of 51 nM in the assay, while all PSBP-6based peptides showed markedly lower inhibitory potencies, with IC50 values ranging from 23 µM to 0.6 mM. Conjugation of 14-mer peptide PSBP-6 with chelator NOTA via an Ava linker resulted in an increase of inhibitory potency by one order of magnitude, from 0.6 mM to 30 µM. Complexation of NOTA-Ava-PSBP-6 with

nat

Cu2+ further

increased inhibitory potency as reflected by the determined IC50 value of 23 µM for nat

Cu-NOTA-Ava-PSBP-6.

18


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Radiosynthesis of

64

Cu-NOTA-Ava-PSBP-6 and lipophilicity. The reaction scheme

for radiolabeling of peptide NOTA-Ava-PSBP-6 with Starting from as little as 2 µg of peptide,

64

Cu is shown in Scheme 2.

64

Cu-NOTA-Ava-PSBP-6 was prepared in

decay-corrected radiochemical yields of greater than 95%. No additional purification step was necessary since the radiopeptide was obtained at high radiochemical purity (>95%) as determined by radio-HPLC analysis. The logD7.4 value of

64

Cu-NOTA-Ava-

PSBP-6 was determined to be -1.6 ± 0.01 (n = 3) which is indicative of the hydrophilic nature of the radiotracer.

((Scheme 2))

Cell binding assay with evaluate binding of

64

Cu-NOTA-Ava-PSBP-6. The cell binding assay used to

64

Cu-NOTA-Ava-PSBP-6 to camptothecin-treated EL4 cells was

based on an in vitro model of cell death that has been previously described.16,30,33 Binding of

64

Cu-NOTA-Ava-PSBP-6 to control and apoptotic EL4 cells over 60 min upon

incubation is shown in Figure 2. The assay was carried out in the absence of Ca2+ (Figure 2a) and in the presence of 2.5 mM Ca2+ (Figure 2b) to confirm previous reports that PSBP-6 binding to PS is Ca2+-independent.

((Figure 2))

A comparable baseline level of

64

Cu-NOTA-Ava-PSBP-6 binding to PS was observed in

control (untreated) cells (Figure 2a and 2b). In the absence of Ca2+, treated EL4 cells showed 1.5-fold higher binding of

64

Cu-NOTA-Ava-PSBP-6 compared to control

(untreated) cells, which was consistent over 60 min of incubation (Figure 2a). After 60 min of incubation, control cells showed uptake of 185 ± 20% radioactivity/mg protein and treated cells showed 286 ± 13% radioactivity/mg protein (n = 3, p

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