Synthesis and functional characterization of novel sialyl LewisX. mimic-decorated liposomes for E-selectin-mediated targeting to inflamed

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1 Supporting Information Synthesis and functional characterization of novel sialyl LewisX mimic-decorated liposomes for E-selectin-mediated targeting to inflamed endothelial cells Chanikarn Chantarasrivong, Akiharu Ueki, Ryutaro hyama, Johan Unga, Shinya Nakamura, Isao Nakanishi, Yuriko Higuchi, Shigeru Kawakami, Hiromune Ando, Akihiro Imamura, Hideharu Ishida, Fumiyoshi Yamashita, Makoto Kiso, Mitsuru Hashida Contents S1. Synthesis of slex mimic-conjugated DSPE-PEG S2. Evaluation of induction effect of TNF-α and IL-1β on E-selectin expression in HUVEC S3. Uptake of liposomes in HUVECS treated or nontreated with TNF-α and IL-1β SI 1

2 S1. Synthesis of slex mimic-conjugated DSPE-PEG Supplementary Scheme S1 SI 2

3 4 1. n Bu 2 Sn/toluene,reflux,5h 2.ethylbromoacetate, n Bu 4 NI/toluene,80 C 3.Ac 2,DMAP/pyridine,RT H 2 C H H H H H H H H H N Et 2 C 6 Bz Ac Bz Ac Ac Bz Bz Ac Ac H N Bz Ac S5 Ac Bz Bz S6 Bz NHTFA NHTFA Na N P n H Supplementary Scheme S2 4.1MNaHaq. MeH, RT 5.DSPE-PEG NHS DMF-H 2 (4:1),RT 49%(5 steps) C 17 H 35 C 17H n Bu 2 Sn/toluene,reflux,5h 2.BrCH(Me)C 2 Et, n Bu 4 NBr/toluene,100 C 3.Ac 2,DMAP/pyridine,RT Me Bz Bz Ac Ac Ac Bz Bz S7 44%(3 steps) NHTFA 1.1MNaHaq. MeH, RT 2.DSPE-PEG NHS DMF-H 2 (4:1),RT 31%(2 steps) H 2 C Me H H H H H H H H H N 7 H N Na N P n H C 17 H 35 C 17H 35 Supplementary Scheme S3 SI 3

4 Supplementary Scheme S4 General procedures: 1 H and 13 C NMR spectra were recorded with a Bruker Avance III 500 spectrometer. Chemical shifts are expressed in ppm (δ) relative to the signal of Me 4 Si as an internal standard. High-resolution mass spectrometry (HRMS) was performed with Bruker Daltonics micrtf (ESI-TF) mass spectrometer. Specific rotations were determined with Horiba SEPA-300 high-sensitivity polarimeter. TLC analysis was performed on Merck TLC (silica gel 60F 254 on glass plate). Compounds were visualized either by exposure to UV light (254 nm) or by soak in a solution of 10% H 2 S 4 in ethanol, a solution of phosphomolybdic acid, H 3 P 4, and H 2 S 4, in H 2, ninhydrin reagent, followed by heating. Column chromatography was performed with the solvent system (v/v) specified on silica gel BW-80S, BW-300 (Fuji Silysia C.), or Wakosil HC-N (Wako Pure Chemical Industries, Ltd.) Gel permeation chromatography on JAIGEL GS-310 (Japan Analytical Industry Co,, Ltd.) was performed with a Japan Analytical Industry Co,, Ltd. LC-9225NEXT. Evaporation and concentration were carried out in vacuo. N-(Benzyloxycarbonyl)-3-aminopropyl (3,4--isopropylidene-β-D-galactopyranosyl)- (1 4)-β-D-glucopyranoside (S1) To a solution of N-Cbz-3-aminopropyl β-d-galactopyranosyl-(1 4)-β-D-glucopyranoside 1 (92.1 mg, 0.17 mmol) in 2,2-dimethoxypropane (1 ml), acetonitrile (1 ml), and acetone (1 ml) was added camphor-10-sulfonic acid (2 mg, mmol), and stirred for 1 d at room temperature under Ar. The reaction mixture was quenched with Et 3 N (12 µl, 0.09 mmol) and concentrated in vacuo. The residue was dissolved with MeH (2 ml) and H 2 SI 4

5 (0.2 ml), and heated for 1.5 h at 80 C. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with EtAc-MeH (95 : 5) to give the title product S1 (62.5 mg, 63%); [α] D 0.8 (c 1.2, CH 3 H); 1 H NMR (CD 3 D):δ (5H, m, Ar), 5.06 (2H, s, PhCH 2 ), 4.35 (1H, d, J 1,2 = 8.4 Hz, H-1 Gal ), 4.27 (1H, d, J 1,2 = 7.9 Hz, H-1 Glc ), 4.19 (1H, dd, J 4,5 = 2.1 Hz, J 3,4 = 5.5 Hz, H-4 Gal ), 4.05 (1H, dd, J 2,3 = 7.2 Hz, H-3 Gal ), (2H, m, H-5 Gal, CH 2 CH 2 ), 3.87 (1H, dd, J 5,6 = 2.5 Hz, J gem = 12.1 Hz, H-6 Glc ), 3.80 (1H, dd, J 5,6 = 4.4 Hz, H-6 Glc ), 3.79 (1H, dd, J 5,6 = 7.8 Hz, J gem = 11.6 Hz, H-6 Gal ), 3.75 (1H, dd, J 5,6 = 4.6 Hz, H-6 Gal ), 3.59 (1H, dt, J = 6.1 Hz, J gem = 9.9 Hz, CH 2 CH 2 ), 3.55 (1H, dd, J 3,4 = 8.8 Hz, J 4,5 = 9.4 Hz, H-4 Glc ), 3.51 (1H, dd, J 2,3 = 8.7 Hz, H-3 Glc ), 3.45 (1H, dd, H-2 Gal ), 3.39 (1H, m, H-5 Glc ), (3H, m, H-2 Glc, CH 2 CH 2 NH), 1.78 (2H, quint, J = 6.4 Hz, CH 2 CH 2 CH 2 ) 1.47 (3H, s, Me), 1.32 (3H, s, Me); 13 C NMR (CDCl 3 ):δ 159.0, 138.5, 129.5, 129.0, 128.8, 111.1, 104.2, 81.0, 80.9, 76.4, 76.4, 75.4, 75.1, 74.9, 74.5, 68.2, 67.4, 62.4, 61.9, 38.8, 30.9, 28.4, 26.5; HRMS: m/z calcd for C 26 H 39 NNa 13 ([M+Na] + ): ; found: N-(Trifluoroacetyl)-3-aminopropyl (3,4--isopropylidene-β-D-galactopyranosyl)-(1 4)- β-d-glucopyranoside (S2) A mixture of N-Cbz-3-aminopropyl compound S1 (631.0 mg, 1.10 mmol) and 5% Pd-C (234.1 mg, 0.11 mmol) in 1,4-dioxane (10 ml) and H 2 (1 ml) was stirred for 6 h at room temperature under H 2 gas. The reaction mixture was filtered by Celite, and concentrated in vacuo. To a solution of the residue and methyl trifluoroacetate (0.55 ml, 5.50 mmol) in MeH (10 ml) was added dropwise Et 3 N (1.50 ml, 11.0 mmol) at room temperature under Ar, and stirred for 4 h. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with EtAc-MeH (80 : 20) to give the title product S2 (523.3 mg, 89%); [α] D 1.3 (c 1.1, CH 3 H); H-NMR (CD 3 D): δ 4.36 (1H, d, J 1,2 = 8.3 Hz, H-1 Gal ), 4.29 (1H, d, J 1,2 = 7.9 Hz, H-1 Glc ), 4.19 (1H, dd, J 4,5 = 2.1 Hz, J 3,4 = 5.5 Hz, H-4 Gal ), 4.04 (1H, dd, J 2,3 = 7.2 Hz, H-3 Gal ), (2H, m, H-5 Gal, CH 2 CH 2 ), 3.88 (1H, dd, J 5,6 = 2.4 Hz, J gem = 12.1 Hz, H-6 Glc ), 3.81 (1H, dd, J 5,6 = 4.3 Hz, H-6 Glc ), 3.79 (1H, dd, J 5,6 = 7.7 Hz, J gem = 11.6 Hz, H-6 Gal ), 3.75 (1H, dd, J 5,6 = 4.5 Hz, H-6 Gal ), 3.62 (1H, dt, J = 6.1 Hz, J gem = 10.1 Hz, CH 2 CH 2 ), 3.55 (1H, dd, J 3,4 = 8.7 Hz, J 4,5 = 9.0 Hz, H-4 Glc ), 3.52 (1H, t, J = 8.7 Hz, H-3 Glc ), (4H, m, H-2 Gal, H-5 Glc, CH 2 CH 2 NH), 3.24 (1H, dd, H-2 Glc ), 1.86 (2H, quint, J = 6.4 Hz, CH 2 CH 2 CH 2 ) 1.47 (3H, s, Me), 1.32 (3H, s, Me); 13 C NMR (CD 3 D):δ (J = 36.7 Hz), 118.7, 111.1, 104.2, 104.2, 81.0, 80.9, 76.4, 76.4, 75.4, 75.1, 74.9, 74.5, 68.1, 62.4, 61.9, 38.2, 29.9, 28.4, 26.5; HRMS: m/z calcd for C 20 H 32 F 3 NNa 12 ([M+Na] + ): ; found: SI 5

6 N-(Trifluoroacetyl)-3-aminopropyl (2,6-di--benzoyl-3,4--isopropylidene-β-Dgalactopyranosyl)-(1 4)-2,6-di--benzoyl-β-D-glucopyranoside (2) To a solution of compound S2 (523.3 mg, 1.07 mmol) in pyridine (8 ml) and toluene (11 ml) was added dropwise benzoyl chloride (1.0 ml, 8.57 mmol) at 0 C under Ar, and stirred for 1.5 h at 0 C. The reaction mixture was quenched with MeH, and concentrated in vacuo. The residue was diluted with EtAc, successively washed with sat. NaHC 3 aq., water, and brine, dried over Na 2 S 4, and concentrated in vacuo. The crude product was chromatographed on silica gel with toluene-etac (83 : : 20) twice to give the title product 2 (651.2 mg, 64%); [α] D 8.1 (c 1.0, CHCl 3 ); 1 H-NMR (CDCl 3 ):δ (4H, m, Ar), (2H, m, Ar), (2H, m, Ar), (5H, m, Ar), 7.36 (1H, m, Ar), (4H, m, Ar), 7.08 (1H, m, NH), 5.37 (1H, t, J = 7.8 Hz, H-2 Gal ), 5.15 (1H, dd, J 1,2 = 8.2 Hz, J 2,3 = 9.5 Hz, H-2 Glc ), 4.87 (1H, dd, J 5,6 = 2.1 Hz, J gem = 12.1 Hz, H-6 Gal ), 4.70 (1H, d, H-1 Gal ), 4.64 (1H, brs, H), 4.54 (1H, d, H-1 Glc ), (3H, m, H-6 Glc, H-6 Gal, H-3 Gal ), (2H, m, H-4 Gal, H-5 Gal ), 4.18 (1H, dd, J 5,6 = 3.7 Hz, J gem = 12.1 Hz, H-6 Glc ), 4.00 (1H, m, H-3 Glc ), 3.79 (1H, ddd, J = 3.9 Hz, J = 7.4 Hz, J gem = 10.0 Hz, CH 2 CH 2 ), (2H, m, H-4 Glc, H-5 Glc ), 3.58 (1H, ddd, J = 4.0 Hz, J = 7.1 Hz, CH 2 CH 2 ), 3.35 (1H, m, CH 2 CH 2 NH), 3.17 (1H, m, CH 2 CH 2 NH), (2H, m, CH 2 CH 2 CH 2 ), 1.64 (3H, s, Me), 1.36 (3H, s, Me); 13 C NMR (CDCl 3 ):δ 166.6, 165.7, 165.5, 165.3, (J = 37.0 Hz), 133.5, 133.3, 129.8, 129.8, 129.8, 129.7, 129.5, 129.5, 129.1, 129.0, 128.5, 128.4, 128.2, (J = Hz), 111.4, 101.6, 100.9, 82.2, 77.3, 73.4, 73.2, 73.2, 72.9, 72.4, 72.2, 67.6, 63.7, 62.1, 37.6, 28.3, 27.6, 26.3, 21.5; HRMS: m/z calcd for C 48 H 48 F 3 NNa 16 [M+Na] + : ; found: N-(Trifluoroacetyl)-3-aminopropyl (2,3,4-tri--acetyl-α-L-fucopyranosyl)-(1 3)- [2,6-di--benzoyl-3,4--isopropylidene-β-D-galactopyranosyl-(1 4)]-2,6-di--benzoyl-β-D-glucopyranosid e (S3) To a mixture of compound 2 (632.0 mg, 0.66 mmol), phenyl 3,4-di--acetyl-2--p-methoxybenzyl-1-thio-β-L-fucopyranoside (458.7 mg, 1.00 mmol), N-iodosuccinimide (268.9 mg, 1.20 mmol), and molecular sieves 4A (3.32 g) in cyclopentyl methyl ether (7 ml) and CH 2 Cl 2 (7 ml) was added dropwise trifluoromethanesulfonic acid (12 µl, 0.13 mmol) at -40 C under Ar, and stirred for 1 d at -40 C. The reaction mixture was quenched with sat. NaHC 3 aq., filtered by Celite, and diluted with EtAc. The organic layer was separated, successively washed with sat. Na 2 S 2 3 aq., water, and brine, dried over Na 2 S 4, and concentrated in vacuo. The crude product was chromatographed on silica gel with toluene-acetone (90 : 10). The resulting mixture was dissolved with CH 2 Cl 2 (20 ml) and H 2 (2 ml). To the solution was added SI 6

7 2,3-dichloro-5,6-dicyanobenzoquinone (334.3 mg, 1.47 mmol), and stirred for 6 h at room temperature. The reaction mixture was diluted with EtAc. The organic layer was separated, successively washed with sat. NaHC 3 aq., water, and brine, dried over Na 2 S 4, and concentrated in vacuo. The crude product was chromatographed on silica gel with toluene-etac (80 : : 25). The resulting mixture was dissolved with pyridine (3.5 ml). To the solution was added acetic anhydride (3.5 ml) and 4-(dimethylamino)pyridine (4.3 mg, mmol), and stirred for 2 h at room temperature under Ar. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with toluene-acetone (90 : 10), and re-chromatographed on silica gel with toluene-etac (80 : 20) to give the title product S3 (396.8 mg, 49%); [α] D (c 1.0, CHCl 3 ); 1 H-NMR (CDCl 3 ):δ (2H, m, Ar), (2H, m, Ar), (2H, m, Ar), (2H, m, Ar), (4H, m, Ar), (2H, m, Ar), (4H, m, Ar), (2H, m, Ar), 7.08 (1H, brt, J = 5.3 Hz, NH), (3H, m, H-3 Fuc, H-4 Fuc, H-1 Fuc ), 5.31 (1H, dd, J 1,2 = 8.1 Hz, J 2,3 = 9.3 Hz, H-2 Glc ), 5.28 (1H, dd, J 2,3 = 7.6 Hz, J 1,2 = 8.4 Hz, H-2 Gal ), 5.16 (1H, q, J = 6.5 Hz, H-5 Fuc ), 5.07 (1H, dd, J 1,2 = 4.1 Hz, J 2,3 = 10.6 Hz, H-2 Fuc ), 4.97 (1H, dd, J 5,6 = 4.3 Hz, J gem = 12.0 Hz, H-6 Gal ), 4.82 (1H, dd, J 5,6 = 8.7 Hz, J gem = 12.0 Hz, H-6 Gal ), 4.67 (1H, dd, J 5,6 = 1.5 Hz, J gem = 12.4 Hz, H-6 Glc ), 4.57 (1H, d, H-1 Gal ), 4.41 (1H, m, H-6 Glc ) 4.41 (1H, d, H-1 Glc ), 4.26 (1H, dd, J 3,4 = 5.3 Hz, H-3 Gal ), 4.21 (1H, dd, J 3,4 = 9.3 Hz, H-3 Glc ), 4.19 (1H, dd, J 4,5 = 2.0 Hz, H-4 Gal ), 4.07 (1H, dd, J 4,5 = 9.6 Hz, H-4 Glc ), 3.90 (1H, m, H-5 Gal ), 3.78 (1H, ddd, J = 3.9 Hz, J = 7.3 Hz, J gem = 9.7 Hz, CH 2 CH 2 ), 3.35 (1H, m, H-5 Glc ), 3.45 (1H, ddd, J = 3.9 Hz, J = 7.4 Hz, CH 2 CH 2 ), 3.35 (1H, m, CH 2 CH 2 NH), 3.07 (1H, m, CH 2 CH 2 NH), 2.12 (3H, s, Ac), 1.86 (3H, s, Ac), 1.84 (3H, s, Ac), 1.72 (1H, m, CH 2 CH 2 CH 2 ), 1.63 (1H, m, CH 2 CH 2 CH 2 ), 1.60 (3H, s, C(CH 3 ) 2 ), 1.34 (3H, d, H-6 Fuc ), 1.34 (3H, s, C(CH 3 ) 2 ); 13 C NMR (CDCl 3 ):δ 170.6, 170.1, 169.6, 166.3, 166.0, 165.5, 164.8, (J = 37.1 Hz), 133.9, 133.7, 133.4, 133.2, 130.3, 129.8, 129.6, 129.3, 129.1, 128.9, 128.8, 128.7, 128.6, (J = Hz), 111.1, 101.2, 100.4, 96.5, 77.4, 74.8, 74.7, 74.2, 73.4, 72.2, 71.6, 68.2, 68.0, 67.2, 64.7, 62.7, 61.8, 37.6, 28.4, 27.8, 26.1, 20.7, 20.6, 20.5, 16.1; HRMS: m/z calcd for C 60 H 64 F 3 NNa 23 [M+Na] + : ; found: N-(Trifluoroacetyl)-3-aminopropyl (2,3,4-tri--acetyl-α-L-fucopyranosyl)-(1 3)-[2,6- di--benzoyl-β-d-galactopyranosyl-(1 4)]-2,6-di--benzoyl-β-D-glucopyranoside (4) A solution of compound S3 (382.5 mg, 0.31 mmol) in 80% trifluoroacetic acid aq. (8 ml) and CH 2 Cl 2 (16 ml) was stirred for 0.5 h at 0 C. The reaction mixture was diluted withe toluene, and concentrated in vacuo. The crude product was chromatographed on silica gel with toluene-acetone (83 : : 25) to give the title product 4 (363.2 mg, 98%); [α] D (c 0.9, CHCl 3 ); 1 H-NMR (CDCl 3 ):δ (4H, m, Ar), (4H, m, Ar), SI 7

8 (12H, m, Ar), 7.06 (1H, m, NH), 5.39 (1H, d, J 3,4 = 2.9 Hz, H-4 Fuc ), (4H, m, H-3 Fuc, H-1 Fuc, H-2 Glc, H-2 Gal ), 5.12 (1H, m, H-5 Fuc ), 5.03 (1H, dd, J 1,2 = 4.0 Hz, J 2,3 = 10.8 Hz, H-2 Fuc ), 4.90 (1H, dd, J 5,6 = 6.9 Hz, J gem = 11.7 Hz, H-6 Gal ), (2H, m, H-6 Gal, H-6 Glc ), 4.71 (1H, d, J 1,2 = 8.0 Hz, H-1 Gal ), 4.41 (1H, dd, J 5,6 = 3.8 Hz, J gem = 12.3 Hz, H-6 Glc ) 4.43 (1H, d, J 1,2 = 8.0 Hz, H-1 Glc ), 4.17 (1H, dd, J 2,3 = 6.6 Hz, J 3,4 = 7.0 Hz, H-3 Glc ), 4.15 (1H, dd, J 4,5 = 6.8 Hz, H-4 Glc ), 4.00 (1H, brt, J 3,4 = J 4,H = 3.2 Hz, H-4 Gal ), 3.79 (1H, ddd, J = 3.8 Hz, J = 7.3 Hz, J gem = 9.7 Hz, CH 2 CH 2 ), 3.72 (1H, td, J 2,3 = J 3,H = 9.0 Hz, H-3 Gal ), 3.65 (1H, dd, J 5,6 = 7.3 Hz, H-5 Gal ), 3.58 (1H, m, H-5 Glc ), 3.49 (1H, m, CH 2 CH 2 ), 3.47 (1H, d, 4-H), 3.36 (1H, m, CH 2 CH 2 NH), 3.09 (1H, m, CH 2 CH 2 NH), 2.11 (3H, s, Ac), 1.91 (3H, s, Ac), 1.87 (3H, s, Ac), 1.74 (1H, m, CH 2 CH 2 CH 2 ), 1.65 (1H, m, CH 2 CH 2 CH 2 ), 1.01 (3H, d, J 5,6 = 6.5 Hz, H-6 Fuc ); 13 C NMR (CDCl 3 ):δ 171.0, 170.5, 170.1, 166.5, 166.0, 165.8, 165.5, (J = 36.7 Hz), 133.8, 133.6, 133.5, 133.4, 130.0, 129.8, 129.6, 129.4, 129.3, 129.2, 129.0, 128.9, 128.7, 128.6, 128.6, 128.2, (J = Hz), 101.2, 100.2, 96.4, 74.8, 74.6, 73.5, 73.5, 73.2, 72.6, 72.5, 71.6, 68.7, 68.1, 67.8, 67.2, 64.8, 62.1, 62.1, 37.5, 28.4, 20.8, 20.6, 20.4, 15.8; HRMS: m/z calcd for C 57 H 60 F 3 NNa 23 [M+Na] + : ; found: N-(Trifluoroacetyl)-3-aminopropyl (2,3,4-tri--acetyl-α-L-fucopyranosyl)-(1 3)-[2,6-di- - benzoyl-3--sulfo-β-d-galactopyranosyl-(1 4)]-2,6-di--benzoyl-β-D-glucopyranoside, sodium salt (S4) A solution of compound 4 (43.8 mg, mmol) and di-n-butyltin oxide (10.1 mg, mmol) in toluene (2 ml) was refluxed with Dean-Stark trap for 5 h under Ar. The reaction mixture was concentrated in vacuo. The residue was dissolved with THF (2 ml) and DMF (0.5 ml). To the solution was added sulfur trioxide trimethylamine complex (10.3 mg, mmol), and stirred for 15 h at room temperature under Ar. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with CHCl 3 -MeH (7 : 1) to give the title product S4 (43.1 mg, 88%); [α] D (c 0.6, CHCl 3 ); 1 H-NMR (DMS-d 6 ):δ 9.14 (1H, t, J = 5.4 Hz, NH), (6H, m, Ar), (14H, m, Ar), (3H, m, H-5 Fuc, H-2 Gal, H-3 Fuc ), 5.24 (1H, d, J 3,4 = 3.2 Hz, H-4 Fuc ), 5.14 (1H, d, J 1,2 = 4.0 Hz, H-1 Fuc ), 5.34 (1H, d, J 4,H = 4.6 Hz, H), 5.05 (1H, dd, J 1,2 = 8.2 Hz, J 2,3 = 9.4 Hz, H-2 Glc ), 4.87 (1H, dd, J 1,2 = 4.0 Hz, J 2,3 = 10.9 Hz, H-2 Fuc ), 4.78 (1H, d, J 1,2 = 8.1 Hz, H-1 Gal ), 4.72 (1H, m, H-6 Gal ), 4.71 (1H, d, H-1 Glc ), 4.57 (1H, dd, J 5,6 = 8.6 Hz, J gem = 11.3 Hz, H-6 Gal ), 4.48 (1H, m, H-6 Glc ), 4.34 (1H, dd, J 5,6 = 4.9 Hz, J gem = 12.2 Hz, H-6 Glc ), 4.29 (1H, dd, J 2,3 = 10.1 Hz, J 3,4 = 2.9 Hz, H-3 Gal ), 4.22 (1H, dd, J 3,4 = 9.5 Hz, H-3 Glc ), 4.19 (1H, m, H-4 Gal ), 3.96 (1H, dd, J 4,5 = 9.6 Hz, H-4 Glc ), 3.78 (1H, m, H-5 Gal ), 3.70 (1H, m, H-5 Glc ), 3.55 (1H, dt, J = 6.2 Hz, J gem = 10.4 Hz, CH 2 CH 2 ), 3.30 (1H, dt, J = 6.6 Hz, CH 2 CH 2 ), (2H, m, CH 2 CH 2 NH), 2.09 (3H, s, Ac), 1.78 (3H, s, Ac), 1.75 (3H, s, Ac), (2H, m, CH 2 CH 2 CH 2 ), 1.24 SI 8

9 (3H, d, J 5,6 = 6.4 Hz, H-6 Fuc ); 13 C NMR (DMS-d 6 ):δ 170.1, 169.3, 169.0, 165.4, 165.1, 164.5, (J = 36.3 Hz), 133.5, 133.3, 133.2, 133.0, 130.2, 129.7, 129.4, 129.3, 129.3, 129.0, 128.9, 128.7, 128.6, 128.4, (J = Hz), 99.9, 99.7, 96.2, 75.7, 75.3, 74.5, 73.7, 72.3, 72.1, 71.3, 70.3, 67.7, 66.7, 66.5, 66.0, 64.2, 62.4, 62.3, 36.1, 28.2, 20.4, 20.3, 20.2, 15.4; HRMS: m/z calcd for C 57 H 59 F 3 N 26 S [M] - : ; found: N-{N-[3-({2-aza-5,7,11-trioxa-6-oxido-1,6,12-trioxo-9-[(1-oxo-octadecyl)oxy]-6-phosphanonacosyl}polyethyleneglycol)propyl]-5-amino-1,5-dioxopentyl}-3-aminopropyl (α-l-fucopyranosyl)-(1 3)-[3--sulfo-β-D-galactopyranosyl-(1 4)]-β-D-glucopyranoside, disodium salt (5) A solution of compound S4 (89.0 mg, mmol) and 1 M NaH aq. (1.35 ml, 1.35 mmol) in MeH (3.5 ml) was stirred for 21 h at room temperature. The reaction mixture was concentrated in vacuo. The residue was dissolved with H 2, and passed through Sep-Pak tc18 (Waters Corporation). The filtrate was purified by gel permeation chromatography on a column of JAIGEL GS310 with H 2. The crude product was dissolved with DMF (1.6 ml) and H 2 (0.4 ml). To the solution was added DSPE-PEG NHS (302.3 mg, 0.10 mmol; SUNBRIGHT DSPE-020GS, NF America Corporation), and stirred for 1 h at room temperature. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with CHCl 3 -MeH (80 : 20) with 1% AcH, CHCl 3 -MeH (80 : 20), and CHCl 3 -MeH-H 2 (56 : 38 : 9). The desired product was treated with ion exchange resin (Dowex 50W-X2 Na + form) using CHCl 3 -MeH (1 : 2) to give the title product 5 (192.4 mg, 80%); 1 H-NMR (CDCl 3 -CD 3 D-D 2 /5 : 5 : 1):δ 5.44 (1H, d, J 1,2 = 4.0 Hz, H-1 Fuc ), 5.23 (1H, m, CH 2 CHCH 2 ), 4.76 (1H, q, J 5,6 = 6.9 Hz, H-5 Fuc ), 4.53 (1H, d, J 1,2 = 7.8 Hz, H-1 Gal ), 4.43 (1H, dd, J = 3.0 Hz, J gem = 12.1 Hz, CHCH 2 C), 4.33 (1H, d, J 1,2 = 8.0 Hz, H-1 Glc ), (4H, m), (9H, m), (194H, m), 3.49 (1H, dd, J 2,3 = 9.1 Hz, H-2 Glc ), 3.45 (1H, m), (6H, m), 2.34 (2H, t, J = 7.6 Hz, C()CH 2 ), 2.32 (2H, t, J = 8.0 Hz, C()CH 2 ), 2.22 (2H, t, J = 7.4 Hz, NC()CH 2 ), 2.21 (2H, t, J = 7.4 Hz, NC()CH 2 ), 1.91 (4H, s), 1.88 (2H, quint, J = 7.4 Hz, C()CH 2 CH 2 CH 2 C()), (4H, m, CH 2 CH 2 CH 2 N), (4H, m, C()CH 2 CH 2 ), (56H, m), 1.19 (3H, d, H-6 Fuc ),0.89 (6H, t, J = 6.9 Hz, CH 2 CH 3 ); ESI-TF: calcd for C 155 H 298 N 3 72 PS [M-2H] 2- : found; SI 9

10 N-{N-[3-({2-aza-5,7,11-trioxa-6-oxido-1,6,12-trioxo-9-[(1-oxo-octadecyl)oxy]-6-phosphanonacosyl}polyethyleneglycol)propyl]-5-amino-1,5-dioxopentyl}-3-aminopropyl (α-lfucopyranosyl)-(1 3)-[3--carboxymethyl-β-D-galactopyranosyl-(1 4)]-β-D-glucopyranoside, sodium salt (6) A solution of compound 4 (157.3 mg, 0.13 mmol) and di-n-butyltin oxide (36.4 mg, 0.15 mmol) in toluene (7 ml) was refluxed with Dean-Stark trap for 5 h under Ar. The reaction mixture was concentrated in vacuo. The residue was dissolved with toluene (3 ml). To the solution were added ethyl bromoacetate (88.5 µl, 0.80 mmol) and tetra-n-butylammonium iodide (49.1 mg, 0.13 mmol), and stirred for 5 h at 80 C under Ar. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with toluene-acetone (93:7 90:10). The solution of the resulting mixture and 4-(dimethylamino)pyridine (1.1 mg, mmol) in Ac 2 (2 ml) and pyridine (2 ml) was stirred for 6 h at room temperature under Ar. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with toluene-acetone (90:10) to give an ethyl ester S5 (29.8 mg, 18%) and a lactone S6 (67.8 mg, 42%). To a solution of S5 and S6 in MeH (4.2 ml) were added 1 M NaH aq. (1.55 ml, 1.55 mmol), and stirred for 18 h at room temperature. The reaction mixture was concentrated in vacuo. The residue was dissolved in H 2, and passed through Sep-Pak tc18 (Waters Corporation). The filtrate was purified by gel permeation chromatography with H 2. The crude product was dissolved with DMF (2 ml) and H 2 (0.5 ml). To the solution was added DSPE-PEG NHS (350.6 mg, 0.12 mmol), and stirred for 1 h at room temperature. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with CHCl 3 -MeH (80 : 20) with 1% AcH, CHCl 3 -MeH (80 : 20), and CHCl 3 -MeH-H 2 (58 : 39 : 3) to give the title product 6 (228.1 mg, 49% in 5 steps); 1 H-NMR (CDCl 3 -CD 3 D-D 2 /5 : 5 : 1):δ 5.43 (1H, d, J 1,2 = 3.3 Hz, H-1 Fuc ), 5.24 (1H, m, CH 2 CHCH 2 ), 4.70 (1H, m, H-5 Fuc ), 4.50 (1H, d, J 1,2 = 7.8 Hz, H-1 Gal ), 4.43 (1H, dd, J = 2.9 Hz, J gem = 12.2 Hz, CHCH 2 C), 4.33 (1H, d, J 1,2 = 7.9 Hz, H-1 Glc ), (5H, m), (9H, m), (206H, m), (8H, m), (4H, m), 3.26 (2H, t, J = 6.9 Hz, CH 2 CH 2 CH 2 N), 2.35 (2H, t, J = 7.5 Hz, C()CH 2 ), 2.33 (2H, t, J = 7.6 Hz, C()CH 2 ), 2.22 (4H, t, J = 7.5 Hz, NC()CH 2 ), 1.93 (4H, s), 1.88 (2H, m, C()CH 2 CH 2 CH 2 C()), (4H, m, CH 2 CH 2 CH 2 N), (4H, m, C()CH 2 CH 2 ), (56H, m), 1.20 (3H, d, H-6 Fuc ),0.89 (6H, t, J = 6.9 Hz, CH 2 CH 3 ); ESI-TF: calcd for C 161 H 308 N 3 73 P [M-2H] 2- : found; Ethyl ester intermediate (S5): 1 H-NMR (CDCl 3 ):δ (4H, m, Ar), (2H, m, Ar), (2H, SI 10

11 m, Ar), (6H, m, Ar), (4H, m, Ar), (2H, m, Ar), 7.04 (1H, m, NH), 5.64 (1H, d, J 3,4 = 3.5 Hz, H-4 Gal ), 5.48 (1H, d, J 3,4 = 2.9 Hz, H-4 Fuc ), 5.39 (1H, dd, J 1,2 = 8.4 Hz, J 2,3 = 9.7 Hz, H-2 Gal ), 5.36 (1H, d, J 1,2 = 4.0 Hz, H-1 Fuc ), (2H, m,, H-2 Glc H-3 Fuc ), 5.20 (1H, brq, J 5,6 = 6.5 Hz, H-5 Fuc ), 4.87 (1H, dd, J 2,3 = 10.9 Hz, H-2 Fuc ), 4.83 (1H, dd, J 5,6 = 8.1 Hz, J gem = 11.7 Hz, H-6 Gal ), 4.74 (1H, d, H-1 Gal ), (2H, m, H-6 Glc, H-6 Gal ), 4.42 (1H, d, J 1,2 = 8.1 Hz, H-1 Glc ), 4.34 (1H, dd, J 5,6 = 3.5 Hz, J gem = 12.4 Hz, H-6 Glc ), 4.20 (1H, dd, J 2,3 = 9.4 Hz, J 3,4 = 9.5 Hz, H-3 Glc ), 4.10 (1H, dd, J 4,5 = 9.7 Hz, H-4 Glc ), 4.06 (2H, s, CH 2 C), (2H, m, CH 2 CH 3 ), (3H, m, H-3 Gal, H-5 Gal, CH 2 CH 2 ), 3.53 (1H, m, H-5 Glc ), 3.47 (1H, ddd, J = 3.9 Hz, J = 7.3 Hz, J gem = 9.8 Hz, CH 2 CH 2 ), 3.35 (1H, m, CH 2 CH 2 NH), 3.07 (1H, m, CH 2 CH 2 NH), 2.13 (3H, s, Ac), 1.89 (3H, s, Ac), 1.87 (3H, s, Ac), (2H, m, CH 2 CH 2 CH 2 ), 1.39 (3H, d, H-6 Fuc ); 13 C NMR (CDCl 3 ):δ 170.8, 170.4, 170.1, 169.9, 169.3, 166.2, 166.0, 165.4, 164.8, (J = 37.1 Hz), 133.9, 133.7, 133.4, 130.0, 129.9, 129.6, 129.3, 129.2, 129.0, 128.9, 128.8, 128.8, 128.6, 128.2, (J = Hz), 101.2, 100.9, 96.4, 78.2, 74.6, 74.5, 73.9, 73.5, 71.7, 71.4, 71.0, 68.5, 67.8, 67.1, 66.8, 65.8, 64.5, 61.7, 61.4, 60.9, 37.5, 28.4, 20.8, 20.7, 20.6, 20.5, 15.9, Lactone intermediate (S6): 1 H-NMR (CDCl 3 ):δ (2H, m, Ar), (2H, m, Ar), (2H, m, Ar), (2H, m, Ar), (6H, m, Ar), (4H, m, Ar), (2H, m, Ar), 7.05 (1H, m, NH), (2H, m, H-2 Gal, H-4 Fuc ), (2H, m, H-3 Fuc, H-1 Fuc ), 5.32 (1H, dd, J 1,2 = 8.0 Hz, J 2,3 = 9.1 Hz,, H-2 Glc ), 5.07 (1H, dd, J 1,2 = 4.1 Hz, J 2,3 = 11.0 Hz, H-2 Fuc ), (2H, m, H-5 Fuc, H-6 Gal ), 4.90 (1H, dd, J 5,6 = 6.2 Hz, J gem = 11.9 Hz, H-6 Gal ), 4.83 (1H, d, J 1,2 = 8.3 Hz, H-1 Gal ), 4.81 (1H, d, J 3,4 = 3.7 Hz, H-4 Gal ), 4.72 (1H, dd, J 5,6 = 1.4 Hz, J gem = 12.4 Hz, H-6 Glc ), 4.61 (1H, d, J gem = 18.5 Hz, CH 2 C), 4.49 (1H, dd, J 5,6 = 3.5 Hz, H-6 Glc ), 4.42 (1H, d, H-1 Glc ), 4.38 (1H, d, CH 2 C), 4.19 (1H, dd, J 3,4 = 9.2 Hz, H-3 Glc ), 4.14 (1H, dd, J 4,5 = 9.4 Hz, H-4 Glc ), 4.04 (1H, dd, J 2,3 = 10.1 Hz, J 3,4 = 3.7 Hz, H-3 Gal ), 3.85 (1H, dd, J 5,6 = 7.6 Hz, H-5 Gal ), 3.80 (1H, ddd, J = 3.9 Hz, J = 7.2 Hz, J gem = 9.7 Hz, CH 2 CH 2 ), 3.58 (1H, m, H-5 Glc ), 3.47 (1H, ddd, J = 3.9 Hz, J = 7.3 Hz, CH 2 CH 2 ), 3.37 (1H, m, CH 2 CH 2 NH), 3.08 (1H, m, CH 2 CH 2 NH), 2.10 (3H, s, Ac), 1.90 (3H, s, Ac), 1.89 (3H, s, Ac), (2H, m, CH 2 CH 2 CH 2 ), 1.23 (3H, d, J 5,6 = 6.6 Hz, H-6 Fuc ); 13 C NMR (CDCl 3 ):δ 170.1, 170.4, 169.8, 166.2, 166.0, 165.4, 165.1, 165.0, (J = 37.1 Hz), 133.9, 133.8, 133.5, 129.9, 129.9, 129.8, 129.6, 129.3, 129.1, 128.9, 128.8, 128.8, 128.6, 128.5, 128.2, (J = Hz), 101.2, 100.3, 96.8, 75.2, 74.6, 74.1, 73.7, 73.3, 71.4, 71.3, 70.6, 68.1, 67.8, 67.1, 66.9, 65.0, 61.9, 60.7, 37.6, 28.4, 21.5, 20.7, 20.6, 20.5, SI 11

12 N-(Trifluoroacetyl)-3-aminopropyl (2,3,4-tri--acetyl-α-L-fucopyranosyl)-(1 3)-[2,6-di- -benzoyl-3--(1s-1-carboxyethyl)- β-d-galactopyranosyl-3,4-lactone-(1 4)]-2,6-di-benzoyl-β-D-glucopyranoside (S7) A solution of compound 4 (100 mg, 84.5 µmol) and di-n-butyltin oxide (23.1 mg, 93.0 µmol) in toluene (8.45 ml) was refluxed with Dean-Stark trap for 6 h under Ar. The reaction mixture was cooled to 100 C. To the solution were added ethyl-2-bromopropionate (220 µl, 1.69 mmol) and tetra-n-butylammonium bromide (27.2 mg, 84.5 µmol), and stirred for 3 h at 100 C under Ar. The reaction mixture was diluted with EtAc, washed with satd aq. NaHC 3, water and brine, dried over Na 2 S 4, and concentrated. The resulting residue was chromatographed on silica gel with toluene-acetone (10:1) to give a mixture of products, which was exposed to high vacuum overnight. The dried residue was then dissolved in pyridine (3.4 ml), and Ac 2 (80 µl) and 4-(dimethylamino)pyridine (catalytic amount) were added to the solution. The reaction mixture was stirred for 3 h at room temperature under Ar and then diluted with EtAc. The solution was washed with 2M HCl, H 2, satd aq NaHC 3 and brine, dried over Na 2 S 4 and concentrated. The crude product was chromatographed on silica gel with toluene-acetone (11:1 6/1) to give S7 (47.1 mg, 44%) and its diastereomer (22.6 mg, 21%); 1 H-NMR (CDCl 3 ):δ (4H, m, Ph), (4H, m, Ph), (2H, m, Ph), (4H, m, Ph), (4H, m, Ph), (2H, m, Ph), 6.99 (1H, bt, J NH,CH2 = 5.4 Hz, NH), 5.49 (1H, d, H-4 Fuc ), (2H, m, H-1 Fuc, H-3 Fuc ), (2H, m, H-2 Gal, H-2 Glc ), (2H, m, H-2 Fuc, H-5 Fuc ), 4.93 (1H, dd, J gem = 11.8 Hz, J 5,6a = 7.4 Hz, H-6a Gal ), 4.84 (1H, dd, J 5,6b = 6.3 Hz, H-6b Gal ), 4.75 (1H, d, H-4 Gal ), 4.67 (1H, d, J 1,2 = 8.2 Hz, H-1 Gal ), 4.58 (1H, dd, J gem = 12.4 Hz, J 5,6a = 1.5 Hz, H-6a Glc ), (3H, m, H-1 Glc, H-6b Glc, CH(Me)C 2 ), (2H, m, H-3 Gal, H-3 Glc ), 4.05 (1H, t, J 3,4 = 4,5 = 9.6 Hz, H-4 Glc ), 3.83 (1H, bt, H-5 Gal ), 3.71 (1H, m, CH 2 CH 2 ), 3.44 (1H, m, H-5 Glc ), 3.39 (1H, m, CH 2 CH 2 ), 3.28 (1H, m, CH 2 CH 2 NH), 3.00 (1H, m, CH 2 CH 2 NH), 2.05 (3H, s, Ac), 1.82 (6H, 2 s, 2 Ac), 1.66 (1H, m, CH 2 CH 2 CH 2 ), 1.56 (1H, m, CH 2 CH 2 CH 2 ), 1.32 (3H, d, CH(Me)C 2 ), 1.24 (3H, d, J 5,6 = 6.6 Hz, H-6 Fuc ); 13 C NMR (125 MHz, CDCl 3 ) δ 170.2, 170.0, 169.8, 169.1, 166.2, 166.0, 165.4, 164.6, 157.4, 157.1, 133.9, 133.7, 133.6, 133.5, 129.9, 129.8, 129.7, 129.6, 129.3, 129.1, 129.0, 128.9, 128.8, 128.8, 128.7, 128.7, 128.6, 128.2, 125.3, 117.0, 114.7, 101.2, 99.9, 96.7, 77.6, 77.3, 77.0, 76.7, 75.1, 74.5, 73.7, 73.3, 72.0, 71.6, 71.4, 71.1, 70.7, 68.2, 67.8, 67.6, 67.1, 65.1, 61.8, 61.1, 37.5, 29.7, 28.3, 21.4, 20.7, 20.7, 20.5, 16.7, 15.4; HRMS (ESI) m/z: found [M+Na] , C 60 H 62 F 3 N 24 calcd for [M+Na] SI 12

13 N-{N-[3-({2-aza-5,7,11-trioxa-6-oxido-1,6,12-trioxo-9-[(1-oxo-octadecyl)oxy]-6-phosphanonacosyl}polyethyleneglycol)propyl]-5-amino-1,5-dioxopentyl}-3-aminopropyl (α-l-fucopyranosyl)-(1 3)-[3--(1S-1-carboxyethyl)-β-D-galactopyranosyl-(1 4)]-β-D- glucopyranoside, sodium salt (7) To a solution of S7 (54.3 mg, 43.9 µmol) in MeH (2.2 ml) was added 1 M NaH aq. (880 µl, 880 µmol), and stirred for 2 h at room temperature. The reaction mixture was concentrated in vacuo. The residue was dissolved in H 2, and passed through Sep-Pak tc18 (Waters Corporation). The filtrate was purified by gel permeation chromatography with H 2. The crude product was dissolved in DMF (660 µl) and H 2 (220 µl). To the solution were added DSPE-PEG NHS (264 mg, 87.8 µmol) and trimethylamine (12,2 µl, 87.8 µmol), and stirred for 1 h at room temperature. The reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with CHCl 3 -MeH (80 : 20) with 1% AcH, CHCl 3 -MeH (80 : 20), and CHCl 3 -MeH-H 2 (58 : 39 : 3) to give the title product 7 (50.2 mg, 31% in 2 steps); 1 H-NMR (CDCl 3 -CD 3 D-D 2 /5 : 5 : 1): δ 5.44 (1H, d, J 1,2 = 3.9 Hz, H-1 Fuc ), 5.22 (1H, m, CH 2 CHCH 2 ), 4.75 (1H, m, H-5 Fuc ), 4.49 (1H, d, J 1,2 = 7.7 Hz, H-1 Gal ), 4.43 (1H, dd, J = 2.9 Hz, J gem = 12.0 Hz, CHCH 2 C), 4.29 (1H, d, J 1,2 = 7.8 Hz, H-1 Glc ), (5H, m), (9H, m), (206H, m), (8H, m), (4H, m), 3.26 (2H, t, J = 6.9 Hz, CH 2 CH 2 CH 2 N), 2.33 (4H, 2t, C()CH 2 ), 2.20 (4H, t, J = 7.3 Hz, NC()CH 2 ), 1.93 (4H, s), 1.88 (2H, m, C()CH 2 CH 2 CH 2 C()), (4H, m, CH 2 CH 2 CH 2 N), 1.59 (4H, m, C()CH 2 CH 2 ), 1.45 (3H, d, J = 6.6 Hz, CH(Me)C 2 ),1.29 (56H, m), 1.19 (3H, d, J = 6.8 Hz, H-6 Fuc ), 0.90 (6H, t, J = 6.8 Hz, CH 2 CH 3 ); ESI-TF: calcd for C 162 H 310 N 3 73 P [M-2H] 2- : found; N-{N-[3-({2-aza-5,7,11-trioxa-6-oxido-1,6,12-trioxo-9-[(1-oxo-octadecyl)oxy]-6-phosphanonacosyl}polyethyleneglycol)propyl]-5-amino-1,5-dioxopentyl}-3-aminopropyl (methyl 5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2 3)-(α-Lfucopyranosyl)-(1 3)-[3-β-D-galactopyranosyl-(1 4)]-β-D-glucopyranoside, disodium salt (native slex-dspe-peg; S9) To a solution of compound S8 (26.8 mg, mmol) in DMF (1.0 ml) and H 2 (1.0 ml) was added SI 13

14 DSPE-PEG NHS (183.6 mg, mmol; SUNBRIGHT DSPE-020GS, NF America Corporation) and CHCl 3 (0.5 ml), and the suspension was evaporated until the suspension turned to clear solution. Then, it was stirred for 2 days at room temperature, and DSPE-PEG NHS (45.9 mg, mmol) was added to the reaction mixture. After stirred for another 1 day, the reaction mixture was concentrated in vacuo. The crude product was chromatographed on silica gel with CHCl 3 -MeH (80 : 20) with 1% AcH, and CHCl 3 -MeH-H 2 (7:4.5:1) to give the title product S9 (62 mg, 54%); ESI-TF: calcd for C 176 H 336 N 5 81 P [M-2H] 2- : found; SI 14

15 S2. Evaluation of induction effect of TNF-α and IL-1β on E-selectin expression in HUVECs HUVECs (Kurabo, saka, Japan) were cultured in a HuMedia-EG2 medium according to the protocol supplied by the manufacturer (Kurabo, saka, Japan). Cells were harvested and suspended in the culture medium, and plated at 400,000 cells/ml in the 12-well plate on the day before experiments. TNF-α (Life Technologies, Carlsbad, CA) and IL-1β (Sigma Aldrich, St. Louis, M) were added together or separately to the medium at a final concentration of 100 ng/ml and 10 ng/ml, respectively. After 5 h, cells were washed with 0.2 ml HEPES buffer (Kurabo, saka, Japan) and incubated with 0.2 ml of % trypsin for 3-5 min at room temperature. The cells were then centrifuged, washed with 2 ml and resuspended with 0.3 ml of ice cold PBS. Thirty microliters of 10µg/mL anti-e-selectin antibody conjugated with FITC (Abcam, Cambridge, UK) was added and cells were incubated on ice for 30 min. The cells were washed 3 times with 2 ml and resuspended in 1 ml of ice cold PBS. Flow cytometry analysis was conducted using a FACSCanto II (BD Biosciences, San Jose, CA) with excitation and emission wavelength settings of 493 and 528 nm, respectively. Ten thousands gated cells were analyzed using fluorescence histogram with the BDFACSDiva software program. Treatment of HUVECs with TNF-α or IL-1β increased the expression of E-selectin on their surface by 6.6 and 7.5 times, respectively, while combined treatment of both of the cytokines resulted in additive induction of expression of the protein (16.9 times). Supplementary Figure S1. Effect of TNF-α and IL-1β on the expression of E-selectin on the surfaces on HUVECs. SI 15

16 S3. Uptake of liposomes in HUVECS treated or nontreated with TNF-α and IL-1β HUVECs were harvested, and suspended in the culture medium, and plated at a density of 100,000 cells/0.5 ml in a 24-well plate on the day before the experiments. Prior to uptake experiments, TNF-α and IL-1β were added to the medium at a final concentration of 100 ng/ml and 10 ng/ml, respectively, and the cells were incubated for another 5 h. At the onset of the uptake experiments, the culture medium was replaced with that containing fluorescein-labeled liposomes (50 nmol total lipid/ml). After incubating for 3 h, the cells were washed with PBS and incubated with 0.3 ml of % trypsin for 3 5 min at room temperature. The cells were then centrifuged and resuspended in 0.2 ml of ice cold PBS. Flow cytometry analysis was conducted using a FACSCanto II (BD Biosciences, San Jose, CA) with excitation and emission wavelengths set of 494 and 519 nm, respectively. Ten thousand gated cells were analyzed using a fluorescence histogram with the BDFACSDiva software program. The cellular uptake of native and mimic slex liposomes on cytokine-treated HUVECs were 2 orders of magnitude greater than nontreated HUVECs, whereas the uptake of PEG liposomes was similar in both cytokine-treated and nontreated cells. Such a big difference is primarily due to augmented expression of E-selectin by treatment with TNF-α and IL-1β (Supplementary Figure S1). The cellular uptake of 3'-CE slex mimic liposomes was also compared in between different ligand densities. The uptake of 3'-CE slex-mimic liposome was only 1.2 times different in between the two concentrations of 2.5% and 5% (Supplementary Figure S2), suggesting that the ligand density of 5% would be high enough. Supplementary Figure S2. Uptake of fluorescein-labeled liposomes for 3 h in HUVECs treated or nontreated with TNF-α and IL-1β. SI 16