Synthesis and Functional Characterization of Novel Sialyl LewisX

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Synthesis and functional characterization of novel sialyl LewisX mimic-decorated liposomes for Eselectin-mediated targeting to inflamed endothelial cells Chanikarn Chantarasrivong, Akiharu Ueki, Ryutaro Ohyama, Johan Unga, Shinya Nakamura, Isao Nakanishi, Yuriko Higuchi, Shigeru Kawakami, Hiromune Ando, Akihiro Imamura, Hideharu Ishida, Fumiyoshi Yamashita, Makoto Kiso, and Mitsuru Hashida Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00982 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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Molecular Pharmaceutics

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Synthesis and functional characterization of novel

2

sialyl LewisX mimic-decorated liposomes for E-

3

selectin-mediated targeting to inflamed endothelial

4

cells

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Chanikarn Chantarasrivong,‡a Akiharu Ueki, ‡ǁb,c Ryutaro Ohyama,b Johan Unga,†a Shinya

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Nakamura,d Isao Nakanishi,d Yuriko Higuchi,a Shigeru Kawakami,§a Hiromune Ando,b,c,e Akihiro

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Imamura,b Hideharu Ishida,b,e Fumiyoshi Yamashita,a Makoto Kiso,b,c,* Mitsuru Hashidaa,c*

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a Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto

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University, 46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8302, Japan.

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b Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu

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501-1193, Japan.

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c Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University,

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Yoshidaushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan

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d Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, 3-4-1

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Kowakae, Higashi-Osaka, Osaka 577-8502, Japan

ACS Paragon Plus Environment

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e Gifu Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu

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University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan.

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‡These authors contributed equally.

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† Present address: Laboratory of Drug and Gene Delivery System, Faculty of Pharma-Sciences,

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Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.

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§ Present address: Department of Pharmaceutical Informatics, Graduate School of Biomedical

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Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki-shi, Nagasaki 852-8588, Japan.

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ǁ Present address: Faculty of Pharmaceutical Sciences, Aomori University, 2-3-1 Kobata,

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Aomori-shi, Aomori 030-0943, Japan.

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*Corresponding Authors: Mitsuru Hashida, Ph.D. Phone: +81-75-753-4525. Fax: +81-75-753-

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4575. E-mail: [email protected]; Makoto Kiso, Ph.D. Phone: +81-58-293-2916.

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Fax: +81-58-293-2918.

E-mail: [email protected]

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ABSTRACT

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Sialyl LewisX (sLeX) is a natural ligand of E-selectin that is overexpressed by inflamed

3

and tumor endothelium. Although sLeX is a potential ligand for drug targeting, synthesis of the

4

tetrasaccharide is complicated with many reaction steps. In this study, structurally simplified

5

novel

6

phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG) for E-selectin-mediated liposomal

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delivery. The sLeX structural simplification strategies include (1) replacement of the Gal-

8

GlcNAc disaccharide unit with lactose to reduce many initial steps and (2) substitution of

9

neuraminic acid with a negatively charged group, i.e., 3'-sulfo, 3'-carboxymethyl (3'-CM), or 3'-

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(1-carboxy)ethyl (3'-CE). While all the liposomes developed were similar in particle size and

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charge, the 3'-CE sLeX mimic liposome demonstrated the highest uptake in inflammatory

12

cytokine-treated human umbilical vein endothelial cells (HUVECs), being even more potent than

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native sLeX-decorated liposomes. Inhibition studies using anti-selectin antibodies revealed that

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their uptake was mediated primarily by overexpressed E-selectin on inflamed HUVECs.

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Molecular dynamics simulations were performed to gain mechanistic insight into the E-selectin

16

binding differences among native and mimic sLeX. The terminally branched methyl group of the

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3'-CE sLeX mimic oriented and faced the bulk hydrophilic solution during E-selectin binding.

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Since this state is entropically unfavorable, the 3'-CE sLeX mimic molecule might be pushed

19

toward the binding pocket of E-selectin by a hydrophobic effect, leading to a higher probability

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of hydrogen-bond formation than native sLeX and the 3'-CM sLeX mimic. This corresponded

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with the fact that the 3'-CE sLeX mimic liposome exhibited much greater uptake than the 3'-CM

22

sLeX mimic liposome.

sLeX

analogs

were

designed

and

linked

with

1,2-distearoyl-sn-glycero-3-


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KEYWORDS: targeted liposomal delivery; inflamed endothelial cell; E-selectin; sialyl LewisX

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mimic; in vitro uptake; molecular dynamics simulation

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FOR TABLE OF CONTENTS USE ONLY

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Title: Synthesis and functional characterization of novel sialyl LewisX mimic-decorated

3

liposomes for E-selectin-mediated targeting to inflamed endothelial cells.

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Authors: Chanikarn Chantarasrivong, Akiharu Ueki, Ryutaro Ohyama, Johan Unga, Shinya

5

Nakamura, Isao Nakanishi, Yuriko Higuchi, Shigeru Kawakami, Hiromune Ando, Akihiro

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Imamura,

Hideharu

Ishida,

Fumiyoshi

Yamashita,

Makoto

Kiso,

Mitsuru

Hashida

7 8 9


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INTRODUCTION

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E-selectin is a transmembrane glycoprotein that is expressed in endothelial cells. Its expression

4

is extremely low in resting endothelial cells, but it is strongly induced via transcriptional

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regulation by inflammatory cytokines such as TNFα and IL-1β.1, 2, 3 The physiological function

6

of E-selectin is to support leukocyte tethering and rolling on specific locations of the

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endothelium and allow for further strong integrin-mediated interactions. Leukocytes recruited at

8

the site of inflamed tissues remove pathogens and cellular debris and produce growth factors for

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tissue repair. Therefore, the expression of E-selectin is involved as an initial trigger in

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attenuating acute inflammatory injury. However, it is also known that the same mechanism

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occurs during the development of tumor tissues.4, 5, 6 Cancer cells and tumor stroma produce

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cytokines to activate the vascular endothelium, recruit and tame innate immune cells, and form a

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robust immunosuppressive network called the tumor microenvironment.

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Augmented expression of E-selectin in endothelial cells at inflamed or tumor tissues is an

15

attractive option for targeted delivery of specific drugs to these tissues. As has been reviewed

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elsewhere,7,

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polymer-based nanoparticles decorated with anti-E-selectin antibodies and small molecular

18

ligands, have been designed and functionally characterized. Studies on immunoliposomes

19

bearing anti-E-selectin antibodies were conducted a relatively long time ago. The first

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demonstration of this concept was undertaken by Bendas et al. in 1998,11 and was later expanded

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by their group and other groups.12, 13, 14, 15 However, sialyl LewisX (sLeX, Neu5Acα2-3Galβ1-

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4(Fucα1-3)GlcNAcβ) tetrasaccharide-decorated nanoparticles have also been studied.16, 17, 18, 19

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sLeX is a naturally occurring E-selectin ligand and is found at the terminus of N- or O-glycans

8, 9, 10

several types of E-selectin-directed drug delivery systems, i.e., lipid- or


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and glycolipids on the surface of leukocytes and tumor cells. Use of such a small molecular

2

ligand is beneficial due to lower immunogenicity compared with the use of immunoliposomes.20,

3

21

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demonstrated to be highly accessible delivery vehicles for E-selectin targeting both in vitro and

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in vivo,22, 23, 24, 25, 26, 27 their availability is hampered by the complexity and difficulty of sLeX

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synthesis. The large-scale production of sLeX is costly, requires considerable technical expertise,

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and involves many synthesis steps.28,

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development of structurally simplified analogs of sLeX (so-called sLeX mimics) is promising.

However, although sLeX-decorated liposomes and nanoparticles have successfully been

29, 30, 31

To overcome these drawbacks, the design and

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The details of sLeX-selectin binding have been investigated in both functional and structural

10

studies. Systematic replacement of the functional groups of sLeX with hydrogen revealed that all

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three OH groups of the Fuc, the 4- and 6-OH groups of the Gal, and the COO– group of the

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NeuAc are required for sLeX to bind to E- and L-selectins.32, 33 In addition, it has also been

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reported that the GlcNAc residue does not play a crucial role in binding. Crystallographic

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analysis of E-selectin and sLeX cocrystals supports the results of functional studies.34, 35, 36 The

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3- and 4-OH groups of the Fuc form a network of interactions with the selectin-bound Ca++ ion

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and several amino acid residues, while the 4- and 6-OH groups of the Gal and the COO– group of

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the NeuAc are responsible for further hydrogen-bond formation. Accordingly, substitution of

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NeuAc with a negatively charged group, such as carboxymethyl, sulfate, and phosphate groups,

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on the 3' position of the Gal would be the most straightforward way to simplify the structure of

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sLeX.37, 38, 39 Taking into account that the GlcNAc residue is not responsible for binding, we

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previously proposed and demonstrated the feasibility of fucosylated 3'-sulfo-lactose as a potent

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and convenient sLeX mimic.40 Replacement of the Gal-GlcNAc disaccharide unit with lactose

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(i.e., Gal-Glc) removes many initial steps.


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Based on reported structure-activity studies of sLeX and its mimics,32, 40, 41, 42 the present study

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was initiated to develop novel glycolipids that are available for E-selectin-targeted drug carriers.

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We conjugated sLeX and its mimics with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-

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polyethyleneglycol-2000 (DSPE-PEG), taking into account that the swollen layers formed with

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PEG chains are required for steric protection of particulate drug carriers.43, 44, 45, 46 In the present

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study, three fucosylated lactose derivatives modified on the 3' position of the lactose unit, i.e., 3'-

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sulfo, 3'-carboxymethyl (3'-CM), and 3'-(1-carboxy)ethyl (3'-CE), were synthesized as sLeX

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mimics. On the basis of previous results of sLeX mimic development,40, 47 we expected that

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fucosylated 3'-sulfo-lactose would be a suitable targeting motif because of its binding affinity to

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E-selectin. The 3'-CM and 3'-CE analogs were newly designed as more chemically stable

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analogs than the 3'-sulfo (Scheme 1). These analogs retain the position of the COO– group of

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NeuAc within sLeX and are expected to engage in hydrogen-bond formation similar to sLeX. In

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addition, the 3’-CE analog was anticipated to provide strong interaction to E-selectin because the

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methyl group of the 3'-CE group limits the free rotation of the COO– group. The most interesting

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finding in this study is that 3'-CE sLeX mimic liposomes, which are newly developed, exhibit

16

greater and more specific uptake than native sLeX in cytokine-activated human umbilical vein

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endothelial cells (HUVECs). The molecular mechanisms underlying the binding of sLeX mimics

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with E-selectin were confirmed by molecular dynamics simulations using the structure of the

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sLeX-E-selectin cocrystal (PDB: 4CSY) as a template.

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EXPERIMENTAL SECTION

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1. Outline of the synthesis of sLeX mimic‐linked phospholipids

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The targeting motifs were synthesized using an efficient, stereoselective route (Scheme 1).

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First, 3-aminopropyl lactoside 1 was prepared according to a previously reported procedure,48

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and it was then converted into the mono-hydroxy form 2 through a four-step reaction sequence.

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Next, compound 2 underwent α-selective fucosylation with donor 3 with the assistance of the

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synergistic solvent effect of CPME-CH2Cl2,49 followed by replacement of the MBn group with

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an acetyl group and the acid hydrolysis of the isopropylidene group at C3' and C4', producing 4.

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3'-OH selective sulfonylation, carboxymethylation and 1-carboxyethylation via tin acetal

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formation and subsequent full deacylation produced 3'-sulfo and 3'-carboxymethyl and 3'-(1-

10

carboxy)ethyl trisaccharides, respectively. Finally, they were conjugated with DSPE-PEG to

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create sLeX mimic targeting motifs 5, 6 and 7, respectively.

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13 14 15 16 17 18

Scheme 1. Synthesis of sLeX mimic-linked phospholipids. (a) CSA/2,2-dimethoxypropane, acetone, MeCN, RT, 1 d, 63%; (b) H2, 5% Pd/C/1,4-dioxane-H2O (2:1), RT, 6 h; (c) TFAOMe, Et3N/MeOH, RT, 4 h, 89% (2 steps); (d) BzCl/toluene-pyridine (4:3), 0°C, 1.5 h, 64%; (e) 3, NIS, TfOH, MS4Å/CPME-CH2Cl2 (1:1), -40°C, 1 d; (f) DDQ/CH2Cl2-H2O (10:1), RT, 6 h; (g) Ac2O/pyridine, RT, 2 h, 49% (3 steps); (h) 80% TFAOH aq./CH2Cl2, 0°C, 0.5 h, 98%; (i) i.


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nBu2SnO/toluene, reflux, 5 h; ii. SO3·NMe3/THF-DMF (4:1), RT, 15 h, 88% (2 steps); iii. 1 M NaOH aq./MeOH, RT, 21 h; iv. DSPE-PEG2000-NHS /DMF-H2O (4:1), RT, 1 h, 80% (2 steps); (j) i. nBu2SnO/toluene, reflux, 5 h; ii. ethyl bromoacetate, nBu4NI/toluene, 80°C, 5 h; iii. Ac2O, DMAP/pyridine, RT, 6 h; iv. 1 M NaOH aq./MeOH, RT, 18 h; v. DSPE-PEG2000-NHS /DMFH2O (4:1), RT, 1 h, 49% (5 steps); (k) i. nBu2SnO/toluene, reflux, 5 h; ii. BrCH(Me)CO2Et, nBu4NBr/toluene, 100°C, 3 h; iii. Ac2O, DMAP/pyridine, RT, 3 h, 44% (2 steps); iv. 1 M NaOH aq./MeOH, RT, 2 h; v. DSPE-PEG2000-NHS, Me3N/DMF-H2O (3:1), RT, 1 h, 31% (2 steps). Cbz = benzyloxycarbonyl; TFA = trifluoroacetyl; Bz = benzoyl; MBn = p-methoxybenzyl; CSA = 10-comphorsulfonic acid; NIS = N-iodosuccinimide; CPME = cyclopentyl methyl ether; DDQ = 2,3-dichloro-5,6-dicyanobenzoquinone; DMF = N,N-dimethylformamide; DMAP = 4dimethylaminopyridine; DSPE-PEG = 1,2-distearoyl-sn-glycero-3-phosphoethanolaminepolyethyleneglycol-2000; NHS = N-hydroxysuccinimidyl.

13 14

Detailed experimental procedures for synthesizing the target compounds and spectroscopic

15

data for structural determination of newly synthesized compounds are described in Supporting

16

Information, section S1.

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2. Preparation of liposomes

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1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Avanti Polar Lipids

20

(Alabaster, AL). Cholesterol, methanol, and chloroform were purchased from Nacalai Tesque

21

(Kyoto, Japan). DSPE-PEG-Fluorescein was prepared through the chemical reaction of DSPE-

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PEG-NH2 (NOF America Corporation, White Plains, NY) and Fluorescein-NHS (Thermo Fisher

23

Scientific, Waltham, MA) following the protocol supplied by the manufacturer. DSPC (2.75

24

µmol/mL), cholesterol (1.95 µmol/mL), native sLeX- or sLeX mimic-linked DSPE-PEG (0.25

25

µmol/mL) and DSPE-PEG-Fluorescein (0.05 µmol/mL) were individually dissolved in

26

chloroform-methanol (1:1). One milliliter of each solution was mixed in a 50-mL round-bottom

27

flask. The solvent of the mixture was removed under reduced pressure using a rotary evaporator.

28

The resultant lipid film was further vacuum-desiccated for at least 6 h. The lipid film was

29

swollen using phosphate-buffered saline (PBS, Nissui Pharmaceutical, Tokyo, Japan) at room


10

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temperature for 30 min and suspended by shaking for 30 min in a water bath at 65°C. The

2

suspension was sonicated in a bath-type sonicator (ASU-3M, AS ONE, Osaka, Japan) at 65°C

3

for 10 min and then a tip-type sonicator (Ultrasonic generator US 300, Nissei, Tokyo, Japan) at

4

an intensity of 200 W for 3 min. The suspension was then extruded 31 times through a 100-nm

5

pore membrane equipped in an extruder (Avanti Mini-Extruder, Avanti Polar Lipids, Alabaster,

6

AL) maintained at 65°C. The liposome solution was purified using a NAP-5 gel filtration column

7

(GE Healthcare, Buckinghamshire, UK) equilibrated with PBS. After the amount of

8

phospholipids in the eluate was determined using a Phospholipids C-test Wako (Wako Pure

9

Chemical Industry), the liposome concentration was adjusted to 500 nmol total lipid/mL. An

10

aliquot of the stock solution was diluted 2.5 times with distilled water, and 750 µL of the

11

solution was taken for measurement of the particle size and zeta potential of the liposomes in a

12

Zetasizer Nano ZS (Malvern, Worcester, UK). Moreover, the fluorescence intensity of each

13

liposome was determined at a total lipid concentration of 1 nmol/mL using a Fluoromax-4

14

spectrofluorometer (Horiba, Kyoto, Japan).

15 16

3. Evaluation of the cellular uptake of liposomes

17

HUVECs were purchased from Kurabo Industry (Osaka, Japan) and cultured according to the

18

protocol supplied by the manufacturer. HuMedia-EG2 (Kurabo, Osaka, Japan) was used as the

19

culture medium. The cells were harvested, suspended in the culture medium, and plated at a

20

density of 100,000 cells/0.5 mL in a 24-well plate on the day before experiments. Prior to uptake

21

experiments, TNF-α (Life Technologies, Carlsbad, CA) and IL-1β (Sigma Aldrich, St. Louis,

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MO) were added to the medium at a final concentration of 100 ng/mL and 10 ng/mL,

23

respectively, and the cells were incubated for another 5 h. At the onset of the uptake


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experiments, the culture medium was replaced with medium containing fluorescein-labeled

2

liposomes (50 nmol total lipid/mL). Following incubation for the indicated time periods, the cells

3

were washed with PBS and incubated with 0.3 mL of 0.0025% trypsin for 3–5 min at room

4

temperature. The cells were then centrifuged and resuspended in 0.2 mL of ice-cold PBS. Flow

5

cytometry analysis was conducted using a FACSCanto II (BD Biosciences, San Jose, CA) with

6

excitation and emission wavelength settings of 494 and 519 nm, respectively. Ten thousand gated

7

cells were analyzed using a fluorescence histogram with the BDFACSDiva software program.

8 9

4. Inhibition of the cellular association of liposomes by anti-selectin antibodies

10

HUVECs were harvested, suspended in the culture medium, and plated at a density of 40,000

11

cells/0.25 mL in a 48-well plate on the day before the experiments. Prior to the experiments, the

12

cells were pretreated with 100 ng/mL TNF-α and 10 ng/mL IL-1β for 5 h. The culture medium

13

was replaced with an equal volume of Hank's buffered salt solution (HBSS) (Nissui

14

Pharmaceutical, Tokyo, Japan) containing one of the anti-selectin antibodies (Abcam,

15

Cambridge, UK) at a concentration of 10 µg/mL, and the cells were then incubated for 30 min on

16

ice. Twenty-five microliters of 500 nmol total lipid/mL liposome solution was added at the onset

17

of the cellular association study. Fifteen minutes later, the cells were washed twice with 0.3 mL

18

of HBSS supplemented with 10% fetal bovine serum (FBS) and once with 0.3 mL of HBSS and

19

then lysed with 0.1 mL of an aqueous lysis buffer composed of 0.05% Triton X-100, 0.075%

20

EDTA, and 1.21% Tris for 15 min on a shaker. The lysates were transferred to a 96-well plate,

21

and their fluorescence intensity was determined using an ARVO MX microplate fluorometer

22

(Perkin Elmer, Waltham, MA) with excitation-emission wavelengths set at 485 and 535 nm,

23

respectively. The protein concentration in 20-µL aliquots of the lysate was measured using the


12

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Protein Quantification Kit-Wide Range (Dojindo Molecular Technologies, Rockville, MD). In

2

addition, the inhibitory effects of anti-selectin antibodies were statistically evaluated using a one-

3

way analysis of variance with Dunnett’s post hoc test.

4 5

5. Confocal fluorescence microscopy analysis of the subcellular distribution of liposomes

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HUVECs were harvested, suspended in the culture medium, and plated at a density of 40,000

7

cells/0.25 mL in an 8-well chamber slide on the day before the experiments. Prior to uptake

8

experiments, TNF-α and IL-1β were added to the medium at a final concentration of 100 ng/mL

9

and 10 ng/mL, respectively, and the cells were incubated for another 5 h. At the onset of the

10

uptake experiments, the culture medium was replaced with medium containing fluorescein-

11

labeled liposomes (50 nmol total lipid/mL). After incubating for 3 h, the cells were washed 3

12

times with PBS and fixed with 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque,

13

Kyoto, Japan). Fifteen minutes later, the cells were washed with PBS, mounted with Vectashield

14

mounting medium containing DAPI (Vector laboratories Inc, Burlingame, CA), and observed

15

using a confocal microscope (Nikon A1RMP/Ti-E/PFS, Nikon Instruments Inc., Melville, NY)

16

equipped with NIS-elements AR 4.13.00 software. The excitation wavelength was set at 488.5

17

nm and 404 nm, and the emission wavelengths were scanned in the range of 500-550 nm and

18

425-475 nm for fluorescence and DAPI, respectively.

19 20

6. Molecular dynamics simulation

21

The structure of the sLeX-E-selectin cocrystal (PDB: 4CSY)50 was used as a template to

22

investigate the molecular binding of sLeX mimics with E-selectin. A molecular dynamics (MD)

23

calculation of the solvated protein-ligand complex was performed using the GROMACS 5.0.4


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package.51 The AMBER-ff99SB force field52 and the AMBER-GAFF53/AM1-BCC54 were

2

assigned for E-selectin and ligand molecules, respectively. Each complex structure was

3

neutralized and placed in a TIP3P55 water box, with a margin of 10 Å between the protein and

4

the boundaries of the periodic box. The particle mesh Ewald method with a cut-off of 8 Å was

5

used to calculate long-range electrostatic interactions. MD simulations of 100 ps for the system

6

relaxation under constant NVT conditions at 300 K and 400 ps for water molecule density

7

equilibration under constant NPT conditions at 1 bar and 300 K were performed before data

8

collection. Subsequently, an MD simulation of 2 ns was performed under constant NVT

9

conditions at 300 K, and the complex structures were extracted in the 201 snapshots taken every

10

10 ps. This MD simulation scheme was replicated three times, and the analyses for hydrogen-

11

bond formations and distributions of dihedral angles were performed in a total of 603 snapshots.

12 13 14

RESULTS

15 16

1. Physicochemical characteristics of native and mimic sLeX liposomes

17

Table 1 summarizes the particle size and zeta potential of the prepared liposomes. The average

18

diameter was similar in all the liposomes, ranging from 96.4 to 106.4 nm. Their polydispersity

19

indices were no more than 0.19, indicating that liposomes with a uniform size distribution were

20

obtained by employing an extrusion method following the hydration-sonication method.

21

Whereas the PEG liposome was nearly neutral with a zeta potential of −6.92 ± 1.23 mV, all

22

liposomes bearing the native or mimic sLeX residues were highly negative with regard to surface

23

charge, but the negative charges associated with native or mimic sLeX were not significantly


14

Page 15 of 37

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1

different. The fluorescence intensities of all prepared liposomes were comparable but, to be more

2

precise, were employed for normalization of cellular uptake.

3 4 5

6 7 8

Table 1. Physicochemical characteristics of native and mimic sLeX liposomes Liposomea)

Average diameter (nm)

PDIb)

Zeta potential (mV)

PEG

102.53 ± 0.46

0.16

–6.92 ± 1.23

Native sLeX-PEG

98.45 ± 1.01

0.16

–24.5 ± 1.1

3'-sulfo sLeX mimic-PEG

106.37 ± 1.46

0.19

–23.97 ± 0.85

3'-CM sLeX mimic-PEG

96.35 ± 0.51

0.17

–23.03 ± 1.7

3'-CE sLeX mimic-PEG

102.33 ± 0.71

0.11

–24.70 ± 1.1

a) PEG, polyethylene glycol; 3'-CM, 3'-carboxymethyl; 3'-CE, 3'-(1-carboxy)ethyl. b) PDI, polydispersity index.

9 10

2. Uptake in cytokine-treated HUVECs

11

Figure 1A shows the time course of the uptake of native and mimic sLeX liposomes in

12

proinflammatory cytokine-treated HUVECs. The cellular uptake of all tested liposomes

13

increased linearly over 4 h. The uptake rate calculated from the slope was highest with the 3'-CE

14

sLeX mimic liposomes (105.2-fold higher than that of the PEG liposome), followed by the 3'-

15

sulfo sLeX mimic (90.1-fold), native sLeX (85.1-fold), and 3'-CM sLeX mimic liposomes (25.5-

16

fold). Hereafter, the cellular uptake of the liposomes was evaluated at 3 h.

17

Figure 1B shows the concentration dependence of the liposome uptake. The uptake of native

18

and mimic sLeX liposomes obeyed nonlinear kinetics within the concentration range tested

19

(0.025–0.2 µmol total lipid/mL). The Eadie–Hofstee plot analysis indicated that the Michaelis–

20

Menten constants (Km) were 0.011, 0.020, 0.025, and 0.057 µmol total lipid/mL for the 3'-CE


15


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Page 16 of 37

1

sLeX mimic, native sLeX, 3'-sulfo sLeX mimic, and 3'-CM sLeX mimic liposomes, respectively

2

(Figure 2). The order of the affinity-related constant values corresponded to that of the uptake

3

rate.

4 5

6 7 8 9 10 11

Figure 1. Time-course analyses (A) and concentration dependence (B) of the uptake of fluorescein-labeled liposomes in HUVECs treated with TNF‐α and IL‐1β for 5 h. Symbols: , PEG liposome; , native sLeX liposome; , 3'-sulfo sLeX mimic liposome; , 3'-CM sLeX mimic liposome; , 3'-CE sLeX mimic liposome.


16

Page 17 of 37

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1 2 3 4 5 6


Figure 2. Eadie–Hofstee diagrams of the uptake of (A) native sLeX, (B) 3'-sulfo sLeX mimic, (C) 3'-CM sLeX mimic, and (D) 3'-CE sLeX mimic liposomes in HUVECs pretreated with 100 ng/mL TNF-α and 10 ng/mL IL-1β for 5 h. The ordinate and abscissa represent the uptake rate (v) and the uptake rate divided by liposome concentration (v/s), respectively. The regressed equation, together with its regression coefficient, is shown in each panel, where


17


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1 2

Page 18 of 37

v v = −Km ⋅ + Vmax s

3

To elucidate involvement of specific uptake processes, the effect of specific antibodies on the

4

cellular association of the liposomes was investigated. Figure 3 shows the cellular association of

5

native and mimic sLeX liposomes in the presence and absence of specific antibodies against E-

6

selectin, P-selectin, or L-selectin (10 µg/mL). The anti-E-selectin antibody inhibited the cellular

7

association of native sLeX, 3'-sulfo sLeX mimic, and 3'-CE sLeX mimic liposomes by 70%,

8

72.7%, and 91.7%, respectively, whereas significant inhibition did not occur for the 3'-CM sLeX

9

mimic liposome. In contrast, neither the anti-P-selectin nor anti-L-selectin antibodies affected the

10

cellular association of any of the liposomes. Therefore, the uptake of native and mimic sLeX

11

liposomes was considered to be mediated mostly by E-selectin, the expression of which was

12

upregulated by pretreatment with TNF-α and IL-1β.

13 14 15

Figure 3. Inhibition by anti‐selectin antibodies of the association of fluorescein‐labeled native and mimic sLeX liposomes with HUVECs treated with TNF‐α and IL‐1β. *P

Synthesis and Functional Characterization of Novel Sialyl LewisX

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