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Title

Preferential capture of EpCAM-expressing

extracellular vesicles on solid surfaces coated

with an aptamer-conjugated zwitterionic polymer.

Author(s)

Alternative

Yoshida, M; Hibino, K; Yamamoto, S; Matsumura, S;

Yajima, Y; Shiba, K

Journal Biotechnology and bioengineering, 115(3): 536-544

URL http://hdl.handle.net/10130/4837

Right

This is the pre-peer reviewed version of the

following article: Biotechnol Bioeng. 2018

Mar;115(3):536-544, which has been published in

final form at https://doi.org/10.1002/bit.26489.

This article may be used for non-commercial

purposes in accordance with Wiley Terms and

Conditions for Use of Self-Archived Versions.

Description

Original Article

Preferential capture of EpCAM-expressing extracellular vesicles on solid surfaces

coated with an aptamer-conjugated zwitterionic polymer.

Mitsutaka Yoshida1, 2, 3

, Kazuhiro Hibino1, Satoshi Yamamoto

1, 2, 3, Sachiko Matsumura

1,

Yasutomo Yajima2, 3

, and Kiyotaka Shiba1, 3

1Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Koto,

Tokyo Japan

2Department of Oral & Maxillofacial Implantology, and

3Division of Oral Implants Research and

Oral Health Science Center, Tokyo Dental College, Tokyo Japan

Correspondence and requests for materials should be addressed to K.S. (email: kshiba@jfcr.or.jp)

1

ABSTRACT

Extracellular vesicles (EVs) collectively represent small vesicles that are secreted from

cells and carry biomolecules (e.g., miRNA, lncRNA, mRNA, proteins, lipids, metabolites,

etc.) that originate in those cells. Body fluids, such as blood and saliva, include large

numbers of EVs, making them potentially a rich source of diagnostic information.

However, these EVs are mixtures of vesicles released from diseased tissues as well as

from normal cells. This heterogeneous nature therefore blurs the clinical information

obtainable from EV-based diagnosis. Here, we synthesized an EpCAM-affinity coating

agent, which consists of a peptide aptamer for EpCAM and a zwitterionic MPC polymer,

and have shown that this conjugate endowed the surfaces of inorganic materials with the

preferential affinity to EpCAM-expressing EVs. This coating agent, designated as

EpiVeta, could be useful as a coating for various diagnostic devices to allow

concentration of cancer-related EVs from heterogeneous EV mixtures.

2

Introduction

The term extracellular vesicles (EVs) has been coined to describe small vesicles found

in extracellular spaces (Lotvall et al. 2014) and are believed to be secreted or broken off

from cells. Depending on the possible mechanisms of their genesis, EVs are given more

specific names, including exosomes and microvesicles, among others (Gould and

Raposo 2013). EVs carry their parental cell’s miRNA (Valadi et al. 2007), mRNA

(Valadi et al. 2007), lncRNA (Sato-Kuwabara et al. 2015), proteins (Hegmans et al.

2004), lipids (Llorente et al. 2013), metabolites (Palomo et al. 2014) and DNA (Balaj et

al. 2011) within or on their membranous particles. They are released from almost all cell

types, and are present in considerable numbers in many body fluids, such as blood,

saliva (Marzesco et al. 2005), urine (Marzesco et al. 2005), mammary secretions (milk)

(Zonneveld et al. 2014), prostatic fluid (Ronquist and Hedstrom 1977), seminal fluid

(Marzesco et al. 2005), cerebrospinal fluid (Chiasserini et al. 2014), ascites (Press et al.

2012), pleural effusion (Roca et al. 2016), bronchoalveolar fluid (Torregrosa Paredes et

al. 2012), aqueous humor (Kang et al. 2014), and pericardial fluid (Beltrami et al. 2017)

and even exhaled breath (Sinha et al. 2013), and they may participate in various cellular

3

activities, including cancer growth (Skog et al. 2008), cancer metastasis (Luga et al.

2012; Peinado et al. 2012), acquisition of drug resistance (Boelens et al. 2014),

infectious disease development (Regev-Rudzki et al. 2013), neurodegeneration (Ritchie

et al. 2013), and immune deficiency (Thery et al. 2002), among others.

The recognition of the prevalence and versatility of EVs now predicts the

emergence of novel medical fields involving EV-based diagnosis (Melo et al. 2015;

Skog et al. 2008), EV-based disease prevention (Bottero et al. 2013) and EV-based

therapeutics (Vader et al. 2014). However, body fluids contain mixtures of EVs and

analysis of these mixtures without further differentiation will therefore give results that

are the averaged features of the mixtures. Exploitation of the potential of EVs as

diagnostic and therapeutic agents will require methodologies that can differentiate EVs

into subgroups based on specific characteristics. Because of this situation, we set our

goal to establish a methodology that would enrich the cancer-related EVs from body

fluids. Here, we report the synthesis of an EpCAM-affinity coating agent, which

consists of a peptide aptamer for EpCAM (which has been used as a hallmark of

circulating tumor cells(Allard et al. 2004; Myung et al. 2010) and a zwitterionic

4

poly-2-methacry loyloxyethyl phosphorylcholine(MPC) polymer. Coating of this

conjugate (designated as EpiVeta ) onto silica or polystyrene surfaces imparted an

affinity for EpCAM on the material’s surfaces. Atomic force microscopy (AFM)

analysis also revealed a preferential binding of EpCAM-positive EVs onto the surface

of an EpiVeta-coated polystyrene plate.

Materials and Methods

Synthesis of EpiVeta. The Ep114 peptide aptamer for EpCAM was conjugated to MPC

copolymer (Lipidure®-5903S, average molecular weight of 400,000, synthesized from a

mixture of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and

2-methacryloyloxyethyl succinate, with mole ratio of 3:6:1 by NOF Corporation,

Tokyo) by a two-step reaction. First, an azido-dPEG7-amine (Quanta BioDesign, Ohio,

USA) was covalently linked to the MPC copolymer by condensation between the amino

group of the linker and the carboxyl group derived from 2-methacryloyloxyethyl

succinate unit of the polymer. This reaction was carried out with 0.5 ml of 0.5% (W/W)

Lipidure-5903S solution in ethanol, 10 equivalent amounts of azido-dPEG7-amine and

50 equivalent amounts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium

5

chloride (DMT-MM) at ambient temperature. After incubation for approximately 12 h,

the mixture was dialyzed in a dialysis tube against methanol for 48 h (MWCO=8,000,

Spectra/Por, Spectrum Laboratories, Houston, USA). After dialysis, the volume of

reaction mixture was reduced to 0.5 mL by evaporation. The modified MPC copolymer

was then conjugated with the peptide [in which the extra C-terminal peptide of

dPEG2-Lys(FITC)-dPEG2-Gly(propargyl)-NH2, or

dPEG2-Ser-dPEG2-Gly(propargyl)-NH2 was appended to Ep114] in the presence of

CuSO4 and sodium ascorbate. The reaction was a click chemistry reaction (Rostovtsev

et al. 2002) between the azide group of the linker and the propargyl group at the

C-terminus of the peptides. For this purpose, the azido-dPEG7-linked MPC copolymer

was mixed with 10 equivalent amounts of peptide in 5 ml of 40% methanol. After

dissolving peptide by sonication, 0.5 M L-ascorbic acid sodium (Wako Pure Chemical

Industries, Osaka, Japan) was added until the reaction mixture turned yellow, followed

by the addition of 0.5 M CuSO4, changing the color to brown. For the click chemistry

reaction, the reaction tube was capped and heated at 50 °C for 30 min in a controlled

microwave synthesizer (Biotage Initiator+, Biotage AB, Uppsala, Sweden). The

6

resultant conjugate was purified by either of the following methods. Method 1

(employed in the experiment shown in Fig. 2 A) consisted of adding the reaction

mixture to ethylenediaminetetraacetic acid (pH 8.0) to 0.5 M, and dialyzing for 1 week

in a dialysis tube with a MWCO=8,000 (Spectra/Por) against 3 L of 50% methanol

containing 0.5 M of ethylenediaminetetraacetic acid (pH 8.0). Further dialysis was

performed by changing the dialysis buffer to 50% methanol. Method 2 (employed in the

experiments shown in Fig. 2 B and 3) involved the use of 1 g of chelating fiber (Chelest

Fiber IRY-HW, Chelest, Osaka, Japan) to remove copper ions. After incubation for 1 h

at ambient temperature, the chelating fiber was removed by filtration through paper.

The conjugate was further purified and concentrated using an ultrafiltration membrane

(Amicon Ultra-15, Merck Millipore, Darmstadt, German) for 50 min and centrifuging at

4,000 × g at ambient temperature. The experiment shown in Fig. 2 A used EpiVeta with

FITC and Fig. 2 B and 3 were EpiVeta without FITC. The estimated efficiency of

peptide incorporation per a EpiVeta molecule was calculated to be 0.11 (Supporting

Information).

Preparation of EpiVeta-coated materials. Silica beads (mean diameter = 75 μm,

7

30511-51, Nacalai Tesque, Kyoto, Japan) were coated with EpiVeta as follows. The

beads were first treated with oxygen plasma at 10 mA for 1 min (SEDE-GE, Meiwafosis,

Tokyo, Japan), and then rinsed once with 100 μL of methanol. The beads were

resuspended in 100 μL of EpiVeta solution, and incubated for 10 h at 4 °C with vigorous

shaking in a PetiSyzer. The suspensions were then transferred to Ultrafree Centrifugal

Filters (Low-binding hydrophilic PTFE membrane 0.45 μm UFC30LH00, Millipore

Corp, MA), and residual EpiVeta was removed by centrifugation at 16,000 x g for 30

seconds at 4 °C (Microcentrifuge 5415R, Eppendorf, Hamburg, German). The beads

were dried and resuspended in 300 μL of PBS by vigorous shaking for 10 h at 4 °C. The

beads were then recovered by centrifuging at 16,000 x g for 30 seconds at 4 °C, and

resuspended in 300 μL of PBS. Similarly, 40 μL of polystyrene bead suspension

(Copolymer Microsphere Suspensions 7545A, mean 42 μm in diameter Thermo Fisher

Scientific Inc. MA, USA) was diluted with 300 μL of ethanol in a Protein LoBIND

plastic tube (Eppendorf). The suspension was dispensed into 2 × 120 μL and the beads

were collected by centrifugation and resuspended in 300 μL of EpiVeta or

Lipidure-5903S solution. The suspensions were vigorously shaken for 1 h at 37 °C in a

8

PetiSyzer and then transferred to Ultrafree Centrifugal Filters and centrifuged 16,000 x

g for 30 seconds at 4 °C (Microcentrifuge 5415R). The beads were then dried

(GCD-051X Ulvac, Miyazaki, Japan) and resuspended in 400 μL of PBS (137 mM

NaCl, 2.68 mM KCl, 8.10 mM Na2HPO4, pH 7.4 buffer ) by vigorous shaking for 1 h at

37 °C. The beads were washed three times with 400 μL of PBS, and the pellets were

resuspended in 400 μL of 1% BSA in PBS by vigorous shaking for 1 h at 37 °C. The

coated beads were incubated with EVs derived from HCT-15 (15 μL of 3.5 × 1010

particles/ mL) for 15 h at 4 °C with shaking. The beads were washed once or twice with

400 μL of PBS, suspended in SDS buffer, and subjected to SDS-PAGE for western blot

analyses.

For AFM experiments (Fig. 3), a 15 × 15 mm polystyrene plate (70090

Heat-Shrinking Pla-Plate, Tamiya, Shizuoka, Japan) was first sonicated in ethanol for 5

min. After removing residual ethanol by forced air blowing, the plate was coated with

40 μL of EpiVeta or Lipidure-5903S and incubated for 16 h at 4 °C. The residual

EpiVeta was removed with a spin-coater (MS-A100, MIKASA, Tokyo, Japan) run at

2,000 rpm for 30 sec. The plate was then air-dried for 30 min, followed by drying in

9

vacuo (GCD-051X) for 2 h. The coated plate was equilibrated with 400 μL of PBS at

4°C for 16 h, followed by washing three times with 400 μL of Milli-Q water. The plate

was attached to a slide glass with glue (Quick 5, Konishi, Osaka, Japan) and air dried. A

10 μL volume of PBS was applied for 1 h at ambient temperature and removed. The

EVs derived from HCT-15 (EpCAM+, 10 μL of 3.5× 10

10 particles/ mL) or HT1080

(EpCAM-, 10 μL of 1.75× 10

11 particles/ mL) were applied for 15 h at 4 °C. After three

washes with PBS, the surface was observed by AFM as described below.

Preparations of EVs. EpCAM-positive (HT-29 and HCT-15) and negative (HEK-293T

and HT-1080) cells were obtained from American Type Culture Collection. HT-29 or

HEK-293T cells were propagated using the BelloCell cell culture system

(CES-BCB01000, Cesco Bioengineering, Taichung) as follows. First, 30 mL of cell

culture (2 × 108

cells) were inoculated into a bottle of BelloCell-500p filled with 120

mL of McCoy’s 5A medium supplemented with 10% fetal bovine serum and

penicillin-streptomycin solution (168-23191, Wako Chemicals, Tokyo, Japan). When

the concentration of the cells reached 1 × 109 cells / 500 mL (approximately 96 h), the

medium was replaced with DMEM/ F12 (B-27 supplement, 20 ng/ mL EGF, 20ng/ mL

10

FGF, PS), and the cells were incubated for an additional 72 h. After the incubation, 125

mL of the supernatant was centrifuged 3,500 x g for 10 min at 4 °C in a JA-14 rotor

(Beckman Coulter, Brea) to remove the cells. The resultant supernatant was filtered

through a Stericup-GP (SCGPT02RE 250 mL 0.22 µm, Polyethersulfone, Merck

Millipore, Darmstadt) using a full Teflon diaphragm vacuum pump (FTP-18A, Iwaki,

Tokyo). The filtered supernatant was then centrifuged at 160,000 × g for 70 min at 4 °C

(L-90K and SW32Ti rotor, Beckman Coulter) to prepare the crude EVs. The sediment

was washed once with 30 mL PBS at 160,000 × g for 70 min at 4 °C, and then

resuspended in 3 mL of 2.5 M sucrose Hepes/NaOH, pH 7.2 buffer. This crude EV

fraction was layered on the top of a 0.25-2 M sucrose, 20 mM Hepes/NaOH, pH 7.2

gradient prepared in a UC tube (Beckman Coulter) with a cylindrical type density

gradient former (4023, Sanplatec, Osaka). The tube was centrifuged at 100,000 × g for

17 h (L-90K, SW32Ti). Ten × 3.3 mL of the fractions were collected from the top of the

tube and their densities were determined with a refractometer (RX-5000α, Atago,

Tokyo). Each fraction was washed by centrifuging at 160,000 × g for 2 h (L-90K,

SW32Ti) in a PC tube (Beckman Coulter) after adding 27 mL of PBS. The pellets were

11

suspended in 300μL (for HT-29) or 500 (for HEK-293T) μL of PBS and transferred to

1.5 mL plastic tubes (MS-4265M, Sumitomo Bakelite, Tokyo). The EVs fractions were

quick-frozen in liquid nitrogen and stored at −70 °C. Characterizations of

density-gradient fractionated EVs are shown in Supporting Information Fig. S1. Similar

EVs were prepared from HCT-15 or HT1080 cells, except that iodixanol was used

instead of sucrose as the gradient medium (Iwai et al. 2016).

Quantification of EVs by NanoSight. The numbers of EVs were determined by

NanoSight (LM10, Malvern Instruments, Worcestershire, UK) (Oosthuyzen et al. 2013).

The instrument was calibrated with silica microspheres 0.10±0.03 μm in diameter (#

24041, Polysciences, Inc., PA, USA). Samples were diluted with PBS buffer to a

concentration of 2–10 × 108 particles/mL, and were measured for 30 seconds at ambient

temperature.

Western blotting. Ten microliters of EV samples were mixed with 8 μL of reducing

sample buffer [1 M Tris-HCl (pH 6.8), 30% glycerol, 6% SDS, 3% 2-mercaptoethanol,

and 0.005% bromophenol blue] and incubated at 95 °C (ALB-121, Sansyo, Tokyo,

Japan) for 5 min. The proteins were then separated by electrophoresis in 10 or 15%

12

polyacrylamide gels (Long Life GEL 10 or 15%, Oriental Instruments, Kanagawa,

Japan) at 1,000 V, 40 mA for 40 min. The separated proteins were then transferred onto

a polyvinylidene difluoride (PVDF) membrane using the iBlot Dry Blotting System

(Invitrogen, CA, USA). Nonspecific binding sites were blocked by incubating the

membrane in Blocking One (03953-95, Nacalai Tesque, Kyoto, Japan) for 1 h, followed

by washing with TBS-T (10 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.05% Tween-20)

three times for 5 min each. The membrane was incubated with anti-EpCAM/VU-1D9

(1:1,000 dilution. GTX-11294, Gene Tex, Irvine, USA) for 1 h in Can Get Signal

Solution 1 (Toyobo, Osaka, Japan), followed by incubation with the secondary antibody

(goat anti-mouse IgG-HRP conjugate, Bio-Rad; 1:2000 dilution) in Can Get Signal

Solution 2 for 30 min in darkness. After three 5-min washes with TBS-T, the signals

were detected using ECL and a ChemiDoc system (Bio-Rad, Hercules, CA, USA).

Immunofluorescence microscopy observations of EpiVeta coated beads. The

immunostaining experiments (Figure 2) were conducted by incubating an aliquot of

beads with 100 μL of 1% BSA in PBS for 60 min at ambient temperature, followed by

three washes with 500 μL of PBS. The beads were then incubated with 1 μg of

13

anti-Ep114 rabbit antibody (prepared by Immuno-Biological Laboratories, Gunma,

Japan, and purified from the serum with a peptide affinity column) or normal rabbit IgG

(sc-3888, Santa Cruz Biotechnology, CA, USA) for 30 min with mild shaking on a

mixer (M-36, Taitec, Koshigaya, Japan). After three washes with 100 μL of PBS, the

rabbit IgGs were visualized by incubating 5 μL of Zenon rabbit IgG labeling reagent

(Component A) for 5 min at ambient temperature in the dark, followed by incubation

with 5 μL of Zenon blocking reagent (Component B) for 5 min at ambient temperature

(Z-25307, Life Technologies). After three washes with 500 μL of PBS, the beads were

observed with a confocal laser scanning microscope (FV1000, Olympus Corporation,

Tokyo, Japan). For the EpCAM binding assay, purified EpCAM (5 μL of 140 ng/ μL)

was incubated with the beads for 60 min at ambient temperature, and then washed three

times with 500 μL of PBS. The bound EpCAM was visualized with 1 μg of

anti-EpCAM antibody (GTX-11294, Gene Tex, Irvine, USA). For the EV binding assay,

the EVs derived from HT-29 (7.7 μL of 6.4× 1011

particles/ mL) and HEK-293T (13.9

μL of 3.5× 1011

particles/ mL) were incubated with the beads for 60 min at ambient

temperature, followed by three washes with 500 μL of PBS. The bound EVs were

14

visualized and with the Zenon labeling system after adding 1 μg of anti-EpCAM

antibody or anti-CD63 antibody (ab8219, Abcam, Cambridge, UK).

AFM observations. MFP-3D (Oxford Instruments, Santa Barbara, USA) was employed

for the observations of EVs on material surfaces under aqueous conditions. Silicon

probes (BL-AC40TS, Olympus, Tokyo, Japan) were used in the tapping mode (Hardij

et al. 2013). Topographic height and phase images of three randomly selected areas

were recorded at 5 × 5 μm2, 512 × 256 pixels, at a scan rate of 0.2 Hz (1 μm/ s). The

SPIP image analyzing software (Image Metrology, Hørsholm, Denmark) was used to

calculate the numbers of particles in the images, with thresholds of 15 nm in height and

2.0 in aspect ratio.

Results and Discussion

Synthesis of EpiVeta. MPC, 2-methacryloyloxyethyl phosphorylcholine has been

designed as a membrane-mimicking polymer unit consisting of methacrylate containing

a phosphoryl-choline polar group (Ishihara et al. 1990; Kadoma et al. 1978). Until now,

various types of MPC-based derivatives have been designed and used as coating agents

that reduce non-specific binding of proteins onto material surfaces (Ishihara et al. 1991;

15

Sawada et al. 2003; Yoneyama et al. 1998). These coating agents usually contain a

zwitterionic MPC unit co-polymerized at various ratios with a hydrophobic polymer

unit, such as n-butyl methacrylate (BMA), to make MPC copolymer. The hydrophobic

unit is responsible for the physical interaction with the surfaces of inorganic materials,

and MPC displaying phosphoryl-choline suppresses non-specific binding of proteins or

cells to various devices (Ishihara et al. 1998; Sawada et al. 2003; Sibarani et al. 2007;

Watanabe and Ishihara 2008; Xu et al. 2010). A unit having a reactive group, such as

p-nitrophenyloxycarbonyl poly(oxyethylene)methacrylate (MEONP) or

2-methacryloyloxyethyl succinate (HOMS), can be doped along with MPC/BMA units,

to allow conjugation of types of biomolecules, such as enzymes and antibodies, to MPC

copolymer via a reactive group (Kim et al. 2012; Sakai-Kato et al. 2004). These

biomolecule-conjugated MPC copolymers can be used for biological functionalization

of the surfaces of medical devices (Watanabe and Ishihara 2008; Xu et al. 2010). In this

study, we aimed to make a coating agent that can endow the surfaces of inorganic

materials with an affinity for EpCAM (Litvinov et al. 1994), one of epithelial markers

expressed on EVs (Taylor and Gercel-Taylor 2008). For this purpose, as shown in

16

Figure 1, we conjugated a MPC copolymer consisting of a 3:6:1 (mole ratio) of

hydrophilic (MPC), hydrophobic (BMA), and reactive (HOMS) units (developed by

NOF Corporation as Lipidure®-5903S), with the peptidic aptamer, Ep114, via a short

polyethylene glycol (PEG) linker, as described in the experimental section. Ep114 is a

12-mer peptide with the sequence Lys-His-Leu-Gln-Cys-Val-Arg-Asn-Ile-Cys-Trp-Ser.

This aptamer has been created as a binder for a recombinant EpCAM molecule using an

in vitro evolution system (the details of the creation of Ep114 will be published

elsewhere), and it can discriminate EpCAM positive cell from negative cell in a

complexed media (an example was demonstrated in Supporting Information Fig. S2).

For conjugation reactions, as well as for tracing the peptide, the Ep114 peptide was

appended with a C-terminal extension, dPEG2-Lys(FITC)-dPEG2-Gly(propargyl)-NH2,

consisting of a fluorescent tag and a propargyl moiety to serve as a reactive group for

the Huisgen reaction(Rostovtsev et al. 2002). Other studies have revealed that flexibility

of the displayed ligands would improve their functionality(Lahiri et al. 1999), so we

also introduced a polyethylene linker. We designated the resulting conjugate as

EpiVeta.

17

Affinity coating of silica and polystyrene beads with EpiVeta. We demonstrated the

capacity of EpiVeta to coat the surfaces of inorganic materials and endow them with an

affinity for EpCAM by coating commercially available silica beads with EpiVeta, as

described in the Methods, and then characterizing the surfaces by immunostaining

experiments (Fig. 2A). Incubation of the EpiVeta-coated beads with anti-Ep114

antibody, raised by immunizing a rabbit by Ep114 peptide (our unpublished results),

revealed retention of the antibodies after washing, whereas control antibodies did not

adhere (Fig.2A a – f), indicating that Ep114 moieties are immobilized and displayed on

the surface of the beads and recognized by the anti-Ep114 antibody. Furthermore, the

coated beads retained purified EpCAM after washing, demonstrating that the beads

have an affinity for EpCAM (Fig. 2A g - i). The beads were then incubated with

EpCAM-positive and EpCAM-negative EVs isolated from HT-29 and HEK-293T cells,

respectively. Beads incubated with EpCAM-positive EVs bound the EVs, as confirmed

by binding of the anti-EpCAM antibody (Fig. 2A j - l). By contrast, beads incubated

with EpCAM-negative EVs generated very little binding signal (Fig. 2A m - o).

Confirmation that the observed binding was from EpCAM expressed on EVs and not

18

free EpCAM molecules in solution was obtained by reacting the EV-incubated beads

with anti-CD63 antibody (as both EpCAM positive and negative EVs express CD63).

Stronger CD63 signals were observed when the beads were incubated with

EpCAM-positive EVs (Fig. 2A p - u), supporting the binding of the EpCAM-positive

EVs to the EpiVeta-coated beads. These experiments were semi-quantitative (the

washing steps after antibody incubation in the EV binding experiments hindered the

quantitative assessments), but the data obtained here confirmed the binding ability of

EpiVeta-coated beads for Ep114. Fluorescence microscopy observations also showed

that the EpiVeta polymer evenly coated the beads (Fig 2A a, d, g, j, m, p and s).

The specificity of the interaction between EpCAM positive EVs and the

EpiVeta-coated surface was confirmed by coating polystyrene beads with EpiVeta or

MPC copolymer, as described in Methods. These beads were then incubated with

EpCAM positive EVs and washed (once or twice), and then the amounts of bound EVs

were evaluated by western blotting using the anti-EpCAM antibody. As shown in

Figure 2B, EpiVeta-coated polystyrene beads retained substantial amounts of EpCAM

after two washings, whereas no bound EpCAM was detected on the MPC-coated beads,

19

even after only one washing. This result confirmed that the EpiVeta-coating was

responsible for the specific interaction of EpCAM-positive EVs with the beads.

AFM evaluation of the interaction between EpiVeta-coated polystyrene and EVs.

The interaction between EpCAM-positive EVs and the EpiVeta-coated surface was

assessed more quantitatively using AFM to count the numbers of bound EVs. In these

experiments, polystyrene plates were coated with EpiVeta or MPC copolymer, as

described in the Methods, followed by incubation with EpCAM-positive or

EpCAM-negative EVs. After washing, the numbers of bound particles were determined

by AFM measurements. The adhesive nature of the material surfaces for biological

molecules allowed substantial numbers of EVs to be observed on non-coated (i.e.,

pristine) polystyrene plates, irrespective of the expression of EpCAM molecules on EVs

(Fig. 3 b and c). This non-specific binding of EVs was markedly reduced by the MPC

coating, indicating that that the zwitterionic polymer can suppress the non-specific

binding of EVs onto inorganic materials, as shown in the case of cells (Holmlin et al.

2001) (Fig. 3 e and f). Coating of the polystyrene plates with EpiVeta resulted in

binding of the EpCAM-positive EVs onto the surface (Fig. 3 h). By contrast,

20

EpCAM-negative EVs did not bind to the surface (Fig. 3 i), validating the EpiVeta

coating as an EpCAM-specific affinity surface. Table 1 summarizes the quantitative

analyses of these AFM observations.

Affinity surfaces have been created on inorganic materials using antibodies conjugated

to self-assembling monolayers (SAMs) (Chen et al. 2003; Herrwerth et al. 2003) as well

as to polymers (Kim et al. 2012). The advantages of peptide aptamers over antibodies as

the source of affinity molecules include the ease of preparation and the versatility in

conjugation to foreign molecules (Shiba 2010). Current technologies allow the chemical

synthesis of large quantities of peptides of 100-residue sizes in a far shorter time than is

needed to obtain antibodies (which must be prepared in biological systems).

Furthermore, various modifications, including conjugations with foreign molecules, can

be incorporated during or after peptide synthesis. If necessary, the affinity of a peptide

can be decreased by rational alteration of the amino acid residues, or it can be increased

by secondary in vitro evolution (Smith and Yu 1996), a process that is generally

difficult to conduct with antibodies. A general disadvantage of peptide aptamers is the

weakness of the affinity when compared to antibodies. However, the “weak but specific”

21

interactions of peptidic aptamers is sufficient for fabricating devices that are required to

continuously separate biomolecules depending on their interactions with material

surfaces, as has been successfully exploited in various types of separation columns. The

aim of this study was to establish a platform system for “a programmable bio-surface”

that would capitalize on the “weak but specific” interactions of peptidic aptamers

toward certain molecules to create affinity surfaces on various materials (Figure S3).

The ability to create peptidic aptamers de novo for specific target molecules by

employing in vitro evolution systems means that bio-surfaces can be created with a

specific affinity for a certain molecule using this method, giving this system a

“programmable” nature.

Acknowledgments

We thank T. Minamisawa, K. Suga, and K. Iwai for the preparations and

characterizations of EVs, and NOF Corporation for providing the MPC polymers.

Author Contributions Statement

22

M. Y. and K. S. wrote the main manuscript text and K. S. and Y. Y. designed overall

experiments. K. H. and S. M. synthesized EpiVeta and M. Y. and S. Y. analyzed the

interaction between EpiVeta and EVs. This work was supported by ‘‘Project for Private

Universities: matching fund subsidy’’ from Ministry of Education, Culture, Sports,

Science and Technology to Y. Y. and a grant from Vehicle Racing Commemorative

Foundation to K. S.

Competing financial interest

K. S. received a joint research funding from NOF Corporation.

23

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Figure legends

Figure 1. Scheme for the synthesis of EpiVeta. MPC-polymer (Lipidure-5903S),

consisting of random zwitterionic (2-methacryloyloxyethyl phosphorylcholine, MPC),

hydrophobic (n-butyl methacrylate, BMA), and reactive (2-methacryloyloxyethyl

succinate, HOMS) units with the ratio of 3:6:1 (average molecular weight of 400,000),

was first ligated with a polyethylene glycol (PEG) linker, azido-dPEG7-amine, by

condensation between the amino group of the linker and the carboxyl group of the

HOMS unit. A peptide with the sequence

Lys-His-Leu-Gln-Cys-Val-Arg-Asn-Ile-Cys-Trp-Ser-dPEG2-Lys(FITC)-dPEG-Gly(pro

pargyl)-NH2 or

Lys-His-Leu-Gln-Cys-Val-Arg-Asn-Ile-Cys-Trp-Ser-dPEG2-Ser-dPEG-Gly(propargyl)-

NH2 at the C-terminus was then conjugated to the modified MPC copolymer by a click

chemistry reaction(Rostovtsev et al. 2002) to synthesize EpiVeta.

Figure 2. Immunological characterizations of EpiVeta-coated beads. (A) Silica

beads (a bright field microscopy image is shown on the left with bar of 100 μm) were

coated with EpiVeta, and their surfaces were observed by confocal laser scanning

29

fluorescence microscopy, after reacting with antibodies or extracellular vesicles (EVs).

Reconstituted 3D images from the fluorescein isothiocyanate (FITC) channel

(representing EpiVeta), the Alexa 594 channel (representing antibodies), and their

merged images are shown. The beads were incubated with Anti-Ep114 antibody (a-c),

normal control antibodies (d-f), purified EpCAM + anti-EpCAM antibody (g-i),

EpCAM+ EVs + anti-EpCAM antibody (j-l), EpCAM

- EVs plus anti-EpCAM antibody

(m-o), EpCAM+ EVs plus anti-CD63 antibody (p-r) and EpCAM

- EVs plus anti-CD63

antibody (p-r). EVs of EpCAM+ and EpCAM

- were obtained from HT-29 and

HEK-293T cells, respectively. (B) Polystyrene beads were coated with EpiVeta (lanes

1-3) or MPC copolymer (lanes 4-6) and incubated with EpCAM+ EVs. After no

washing (lanes 1 and 4), or washing once (lanes 2 and 5) or twice (lanes 3 and 6), the

amounts of EpCAM (EpCAM+ EVs) molecules retained on the beads were evaluated by

western blotting. HCT-15 EVs were used for EpCAM+ EVs.

Figure 3. AFM observations of EVs on EpiVeta-coated polystyrene surfaces. The

panels show 3D images of the surfaces of non-coated (upper), MPC copolymer coated

(middle), and EpiVeta-coated (lower) polystyrene plates. The left, middle, and right

30

panels show the results obtained from the surfaces incubated with no EVs or with

EpCAM+ EVs (HCT-15 EVs) and EpCAM

- EVs (HT-1080 EVs), respectively. The

colored bar represents the height of the particles. Representative images from three data

are shown for each sample.

Table 1 Summary of numbers on particles (EVs) observed by AFM on the surfaces of

pristine, MPC copolymer-coated, and EpiVeta-coated polystyrene (PS) plates.

MPC copolymer EpiVetaMPC unit Ep114

FITC

azido-PEG7-amineEp114-PEG2-K(FITC) PEG2-G(propargyl)-NH2

BMA unit

HOMS unit

azideazide

anti-Ep114 Ab(Alexa 594)

cont Ab(Alexa 594)

EpCAM+EpCAM Ab(Alexa 594)

Silica beadsEpiVeta (FITC)

a b c

d e f

g h i

j k l

m n o

p q r

s t u

EpiVeta (FITC) Abs (Alexa 594) Merged

Bar: 100 µm

EpCAM+ EVs+EpCAM Ab(Alexa 594)

EpCAM- EVs+EpCAM Ab(Alexa 594)

EpCAM- EVs+CD63 Ab(Alexa 594)

EpCAM+ EVs+CD63 Ab(Alexa 594)

A

B

1 2 3 4 5 6

0 1 2 0 1 2

Input

EpCA

M

EpiVeta MPC copolymerpolymerwash

no EVs EpCAM+ EpCAM- no

neEp

iVet

aM

PC c

opol

ymer

30nm

15

0

-15

-30

Figure 3

a b c

d e f

g h i

Supporting Information

Preferential capture of EpCAM-expressing extracellular vesicles on solid surfaces

coated with an aptamer-conjugated zwitterionic polymer.

Mitsutaka Yoshida1, 2

, Kazuhiro Hibino1, Satoshi Yamamoto

1, 2, Sachiko Matsumura

1,

Yasutomo Yajima2, and Kiyotaka Shiba

1

1Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Koto,

Tokyo Japan

2Department of Oral & Maxillofacial Implantology, Division of Oral Implants Research and Oral

Health Science Center, Tokyo Dental College, Tokyo Japan

Supporting Materials and Methods.

Estimation of incorporation efficiency of Ep114 in EpiVeta. Numbers of Ep114

incorporated in a EpiVeta molecule were calculated as follows. First, a standard

concentration curve of fluorescein isothiocyanate was obtained by measuring

absorbance at 495 nm, which was used to estimate numbers of FITC molecules in

EpiVeta solution. Similarly, a standard concentration curve of Lipidure®-5903S was

deduced by measuring absorbance at 800 nm, in which observed density, average

molecular weight, i.e., 400,000 and molecular ratio of HOMS, i.e., 0.1 of

Lipidure®

-5903S were used for calculation. The effeminacy of peptide incorporation of

0.11 was obtained from measuring absorbance at 495 nm and 800 nm of EpiVeta

solution.

Supporting Results.

Figure S1. Characterizations of density-gradient fractionated extracellular vesicles

(EVs). EVs from the conditioned media from HT-29 and HEK-293T were separated

into 10 fractions by sucrose gradient centrifugation, as described in the Methods. A.

Fractions indicated in the left from HT-29 were observed by atomic force microscope

(AFM, MFP-3D, Oxford Instruments, Santa Barbara, USA) in a buffer. Bars represent

500 nm. B. Results obtained from nanoparticle tracking analysis (NTA) analyses from

HT-29. The abscissa axis indicates diameters of particles and the vertical axis represent

the numbers of particles calculated. Numbers in the figures show the calculated

concentrations of total particles in the fraction. C. Western blotting and silver staining

(by SilverQuest, Invitrogen, CA, USA) of each fraction from HT-29 and HEK-293T

cells. Sucrose densities in each fraction are shown schematically at the top. Numbers on

the right indicate the molecular weight × 10-3

of standards. Antibodies used in these

experiments and their dilution ratios were mouse anti-EpCAM/TROP-1 (AF960, R&D

Systems, MN; 1:1,000), rabbit anti-CD44 (HPA005785, Sigma, Diegem; 1:1,000),

mouse anti-CD9 (ab124476, Abcam, Cambridge; 1:500), mouse anti-CD63 (ab8219,

Abcam; 1:1,000), mouse anti-Alix (634501, BioLegend, CA; 1:500), rabbit

anti-Annexin V (ab-48815, Abcam; 1:1600), goat anti-rabbit IgG (H+L)-HRP conjugate

(#170-6515, Bio-Rad, USA; 1:2000), and goat anti-mouse IgG (H+L)-HRP conjugate

(#170-6516, Bio-Rad; 1:2000).

Figure S2. Cell staining by Ep114-FITC. A, EpCAM expressing HT-29 cells were

grown in RPMI 1640 (Thermo Fisher Scientific, Waltham, USA) supplemented with

10% of fetal bovine serum, in which Ep114-FITC was added to 0.5 µM. Observations

by fluorescence microscope (two views were shown) indicated the surface staining by

Ep114-FITC (green signals). DAPI was used to stain nuclei. Note that no washing step

was included in the experiment. B, Similar experiment was performed with

EpCAM-non-expressing cell line, HT1080.

Figure S3. Surface coating of various materials with EpiVeta. A solution (50 μL) of

EpiVeta was applied to pieces of the various materials (10 x 10 mm) and incubated for

15 h at 4 °C. The residual EpiVeta was then removed on a spin-coater (MS-A100,

MIKASA, Tokyo, Japan) at 2,000 rpm for 30 sec. The coated materials were air dried

for 30 min, and then dried in vacuo (GCD-051X, Ulvac, Miyazaki, Japan) for 2 h. After

equilibration in PBS for 30 min, the Ep114 immobilized on the materials was detected

using rabbit anti-Ep114 antibody. Primary antibodies were probed with horse radish

peroxidase (HRP)-conjugated secondary antibodies and detected using enhanced

chemiluminescence (ECL) and a ChemiDoc system (Bio-Rad, Hercules, CA, USA).

Control experiments were performed on non-coated (none) and poly-MPC

(Lipidure-5903S) pieces. Non-immune antibody was also used for control experiments.

All materials investigated produced fluorescence and stained with anti-Ep114 antibody

after EpiVeta coating, indicating that EpiVeta can immobilize Ep114 peptide onto the

surfaces of PDMS (polydimethylsiloxane), polystyrene (PS, styrene123-13), silicon

wafers (Si, Panasonic, Osaka, Japan), polyethylene terephthalate (PET, Acrysunday,

Kashiwa, Chiba), polypropylene (PP, PF-11 natural, Acrysunday), polycarbonate (PC,

PC3045 × 03clear), and polyvinyl chloride (PVC, Sawada Plastic, Japan). Under the

conditions used, only Teflon did not show any signal, suggesting it did not become

coated with EpiVeta (data not shown). For PDMS, the effect of a hydrophilic treatment

was also tested. For this purpose, pieces of PDMS were treated with oxygen plasma 20

Pa, 20 sccm, 75 W, for 5 sec using a reactive ion etching system (RIE-10NR, Samco

international, Kyoto, Japan). This oxygen plasma-treated PDMS is denoted as PDMS*

in the figure. In this experiment, EpiVeta having the Ep114 peptide with the C-terminal

extension of GlyGly-Lys(FITC)-Gly-Gly(propargyl)-NH2 was used.