Comparative binding and uptake of liposomes decorated with mannose oligosaccharides by cells expressing the mannose receptor or DC-SIGN

Article abstract of DOI:10.1016/j.carres.2019.107877

Mannose Receptor (MR) and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) are two mannose-specific targets for antigens carried by liposomes but DC-SIGN is more specific of DCs. Here, DC targeting is addressed by using DPPC/D

Full text of DOI:10.1016/j.carres.2019.107877

Journal Pre-proof
Comparative binding and uptake of liposomes decorated with mannose
oligosaccharides by cells expressing the mannose receptor or DC-SIGN
Haifei Gao, Cristine Gonçalves, Téo Gallego, Marc François-Heude, Virginie Malard,
Véronique Mateo, François Lemoine, Virginie Cendret, Florence Djedaini-Pilard,
Vincent Moreau, Chantal Pichon, Patrick Midoux
PII:
S0008-6215(19)30510-5
DOI:
https://doi.org/10.1016/j.carres.2019.107877
CAR 107877
Reference:
To appear in: Carbohydrate Research
Received Date: 27 August 2019
Revised Date: 8 November 2019
Accepted Date: 11 November 2019
Please cite this article as: H. Gao, C. Gonçalves, Té. Gallego, M. François-Heude, V. Malard, Vé.
Mateo, Franç. Lemoine, V. Cendret, F. Djedaini-Pilard, V. Moreau, C. Pichon, P. Midoux, Comparative
binding and uptake of liposomes decorated with mannose oligosaccharides by cells expressing
the mannose receptor or DC-SIGN, Carbohydrate Research (2019), doi: https://doi.org/10.1016/
j.carres.2019.107877.
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Comparative Binding and Uptake of liposomes decorated with mannose
oligosaccharides by cells expressing the mannose receptor or DC-SIGN
Haifei Gao1, Cristine Gonçalves1, Téo Gallego2, Marc François-Heude2, Virginie Malard1,
Véronique Mateo3, François Lemoine3, Virginie Cendret2, Florence Djedaini-Pilard2, Vincent
Moreau2 and Chantal Pichon1* and Patrick Midoux1*
1Centre de Biophysique Moléculaire, CNRS UPR4301, Inserm and University of Orléans, F-
45071, Orléans cedex 02, France.
2Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources, LG2A UMR7378,
ICP FR 3085, Université de Picardie Jules Verne, 80039 Amiens Cedex.
3Sorbonne University, UMR-S INSERM U1135, CNRS ERL 8255, Centre d'Immunologie et
Maladies Infectieuses (CIMI-Paris), Paris, France.
*to whom correspondence should be addressed: Patrick.midoux@cnrs-orleans.fr;
Chantal.pichon@cnrs.fr
ORCID
Florence Djedaini-Pilard: 0000-0002-2841-0023
Patrick Midoux: 0000-0002-8762-366X
Key words: oligosaccharides; glycolipids; liposomes; dendritic cells; Mannose Receptor;
DC-SIGN
1

Abstract
Mannose Receptor (MR) and DC-specific intercellular adhesion molecule-3-grabbing non-
integrin (DC-SIGN) are two mannose-specific targets for antigens carried by liposomes but
DC-SIGN is more specific of DCs. Here, DC targeting is addressed by using DPPC/DOPE
liposomes decorated with a series of diether lipids with a polar head of either a mannose
(Man), tri-antenna of α-D-mannopyranoside (Tri-Man), [Manα1-3(Manα1-6)Man] (Man-tri),
pseudo-Man4 (PMan4) or pseudo-Man5 (PMan5). Liposomes decorated with Man-Tri show the
highest binding and internalization in cells expressing DC-SIGN and in human monocytes-
derived DCs. Conversely, cells expressing MR bind and take up Tri-Man liposomes 3-fold
higher than Man-tri liposomes. Comparatively, liposomes decorated with PMan4 and PMan5
do not show any advantages. Overall, the results indicate that liposomes decorated with Man-
tri residues are more selective toward DCs than those with Tri-Man thanks to better
recognition by DC-SIGN.
2

1. Introduction
Dendritic cells (DCs) are the most powerful professional antigen presenting cells for cancer
immunotherapy by inducing cytotoxic CD8+ T lymphocytes against tumor cells [1-4].
Nowadays, antigens can be proteins, peptides, DNA or mRNA [5-12]. However, a selective
transport of antigens is required to favor their intracellular delivery in DCs in vivo. For this
purpose, liposomes are powerful carriers and their decoration with ligands recognizing
specific receptors on DC surface can improve the targeting and endocytosis of antigens.
Carbohydrate-based targeting is an obvious manner to enhance the capture and uptake by DCs
of antigen pay loaded liposomes because DCs express several sugar receptors binding
oligosaccharides [13-16]. The Mannose Receptor (MR) (CD206) and the DC-specific
intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) (CD209) induce a
clathrin-dependent mediated endocytosis of terminal “high-mannose oligosaccharides”. In
contrast to MR expressed also in various kind of cells including monocytes, macrophages,
subsets of endothelial cells, retinal pigment epithelium, kidney mesangial cells, and tracheal
smooth muscle cells, DC-SIGN is only expressed by DCs [17]. In humans, DCs in mucosal
sites, skin and lymph nodes express preponderantly DC-SIGN [18]. It is not expressed by
monocytes, activated monocytes, T cells, activated T cells, B cells, activated B cells and
CD34+ bone marrow cells. DC-SIGN expressing cells are present in the T cell area of lymph
nodes, tonsils and spleens. In skin, dermal DCs express DC-SIGN but not Langerhans cells
3

nor macrophages. Mature DCs only express DC-SIGN allowing the endocytosis of antigens
and their presentation by the MHC type I and II [18-21]. In blood, plasmacytoid DCs do not
express DC-SIGN while myeloid DCs do. DC-SIGN plays an important role in DCs adhesion
to naive T lymphocytes and endothelium of blood and lymphatic vessels, in their migration, in
inflammation, in the activation of primary T cells, and in triggering of the immune response
[22]. While MR recognizes mannose located at the end of the carbohydrate chains, DC-SIGN
preferentially binds to branched mannose [16]. DC-SIGN recognizes also fucosylated glycans
such as the blood-type Lewis antigens (Lea, Leb, LeX, LeY, and sulfo-Lea) [23]. Liposomes
decorated with Lewis antigens were used to deliver tumor antigens and induce anti-tumour T
cell responses [24, 25]. Thus, targeting this receptor would both increase uptake and reduce
the antigen dilution after in vivo administration. α-D-mannopyranosyl derivatives are usually
used because they are available under forms that can be easily conjugated to various carriers
but the binding affinity typically is weak (K ~ 10-3 M – 10-4 M). The multivalent decoration of
proteins, polymers or liposomes with monosaccharide units increases the apparent affinity by
1 or 2 orders of magnitude [26, 27]. High-mannose oligosaccharides such as Man9 and Man8
exhibiting high affinity (K = 10-6 M) for membrane lectins should be more attractive targeting
ligands, but their synthesis is hard, expensive and yields are weak. Therefore, pseudo-Man9
(PMan9), pseudo-Man8 (PMan8), pseudo-Man5 (PMan5) and pseudo-Man4 (PMan4) mimicking
the High-mannose oligosaccharides or moieties were synthetized in which triazole groups
4

replaced some mannopyranosyl residues [28, 29]. PMan4 mimics the Manα1-2Manα1-
2Manα1-3Man tetrasaccharide (D1 arm) of Man9; PMan5 the Manα1-2Manα1-6 (Manα1-
2Manα1-3)Manα1- pentasaccharide (D2 and D3 arms) of Man9 and Man8; Man-tri the
Manα1-6(Manα1-3)Manα1- trisaccharide (D2 and D3 arms) of Man5, Man6 and Man7
(Supplementary Figure 1). Interestingly, affinities of the same order of magnitude against
lectins were observed for pseudo-Mann/Mann pairs in the case of the higher homologues
supporting their functional equivalency. The relative affinity of PMan9, PMan8 and PMan5
was ~2-fold lower than Man9, Man8 and Man5, respectively [29]. Compared to the methy-α-
D-mannoside (MeαMan) the relative affinity of PMan5, PMan4 and Man-tri with
Concanavalin A (ConA) was 20.8-, 3.8-, and 11-fold higher, respectively and with the
recombinant human macrophage mannose receptor it was 30.6-, 2.12- and 10-fold higher,
respectively [29].
In this work, we synthetized a series of glycolipids comprising either mannose, a tri-antenna
of α-D-mannopyranoside, the [Manα1-3(Manα1-6)Man], the pseudo-Man4 or the pseudo-
Man5. Next, we compared the binding and uptake of a series of fluorescein-labelled liposomes
decorated with these glycolipids by DC2.4 cells expressing the mannose receptor, DC-SIGN
HEK293T cells expressing DC-SIGN and human monocytes derived DCs (hMo-DCs).
2. Results and discussion
2.1. Synthesis of Mannosylated lipids and liposomes preparation
5

A series of new glycolipids (2-7) similar to the Tri-Man diether lipid 1 (Figure 1A) was
synthetized comprising an archaeal diether lipid linked via an oligoethylene spacer to one of
the mannose structures (Figure 1B) [30]. Archaeal lipids exhibiting adjuvant properties
independent on Toll-like receptors activation could be benefit to boost the immune response
[31-33]. Thus, various azido glycosides (2a-7a) were linked by “click chemistry” to the
diether lipid with more than 95% of yield leading to glyco-diether lipids 2-7. For this purpose,
the propargyl-PEG8-diether was synthetized (Figure 2).
Then, fluorescein-labelled liposomes (Flu-Lip) were prepared comprising DPPC (47.25%),
DOPE (47.25%), one glyco-diether lipid (5%) and Flu-DOPE (0.5%) (Supplementary Figure
2). The corresponding liposomes (1b-7b) exhibited a size of ~150 nm in diameter and a
negative ζ potential varying with the sugar polar head nature (Table I).
2.2. Binding to mannose specific lectins
The binding of the liposomes to ConA and BC2LA was evaluated as a function of their
oligomannose structures. The Flu-Lip equipped with Tri-Man (1), Man-tri (4), PMan4 (6) and
PMan5 (7) showed better and similar apparent binding to ConA than with Man (3) (Figure
3A).
6

The binding profile was similar with BC2LA excepted that the binding of Man-tri-spa (5b)
was lower than Man-tri (4b) (Figure 3B). There was a weak binding of Glc (2b) due to a
small affinity of ConA for α-D-glucosyl residues. In contrast, Glc (2b) did not interact with
BC2LA. Thus, those oligomannose structures conserved their binding capacity to lectins after
their linkage to the diether lipid and insertion in the lipid bilayer of liposomes. The results
were in agreement with data reported in literature indicating that compared to MeαMan, the
relative affinity to ConA of a tri-antenna mannopyranoside, PMan5 (7a), PMan4 (6a) and
Man-tri (4a) was 16-, 20.8-, 3.8- and 11-fold, respectively [29]. Here, the binding of
liposomes PMan4 (6b) was similar to PMan5 (7b).
2.3. Binding and uptake of liposomes by DC2.4 cells and DC-SIGN expressing HEK293 cells
DC2.4 cells express the MR while DC-SIGN HEK293 cells express only DC-SIGN
(Supplementary Figure 3). Flow cytometry was used to assess the binding and uptake of Flu-
Lip by these cells and analyses were performed as described in Supplementary Figure 4. The
mean of the fluorescence intensity (MFI) of cells incubated with Flu-Lip was first measured
without any treatment (NT), the second time in the presence of Trypan blue (TB) and the third
time in the presence of monensin (MO). The MFI decrease upon TB treatment quenching
fluorescein fluorescence on the cell surface indicated that ~ 70% of liposomes associated to
the cells were bound on the cell surface; the remaining MFI came from the fluorescence of
intracellular liposomes. The MFI increase upon MO treatment raising the quenching of the
7

fluorescein fluorescence in acid intracellular compartments indicated that liposomes trafficked
through endosomes and lysosomes.
This series of MFI measurements allowed determination of the amount of liposomes bound
and internalized by the cells and thanks to the MO treatment the type of uptake. On this base,
the cell surface binding upon 3h incubation at 37°C was determined from MFI measured
without any treatment minus MFI measured in the presence of TB. Of note: cells were
harvested without trypsin in order to prevent harvesting of liposomes bound on the cell
surface.
The intracellular quantity of liposomes was determined from MFI measured upon monensin
treatment - which rose the fluorescein fluorescence quenched in acid intracellular
compartments such as endosomes and lysosomes - minus fluorescence (MFI-MFITB)
corresponding to liposomes bound on the cell surface. Moreover, increased MFI upon
monensin treatment meant that liposomes uptake likely occurred through acidic compartments
such as endosomes and lysosomes, presumably via clathrin-dependent endocytosis. Although,
Tri-Man (1b) bound more on the surface of DC2.4 cells, their intracellular quantity was close
to that observed with Man (3b), Man-tri-spa (5b) and PMan4 (6b) (Figure 4Ba).
In cells expressing DC-SIGN, the highest binding and internalization was observed with Man-
tri (4b). The uptake was 2- to 3-fold higher than with Tri-Man (1b), PMan4 (6b) and PMan5
(7b) (Figure 4Bb). The monensin enhancement was lower (< 2.5) in DC2.4 cells (Figure 4Ca)
8

than in DC-SIGN HEK293 cells (> 4) (Figure 4Cb). In the latter cells, this enhancement
increased with liposomes concentration indicating that liposomes accumulated in acidic
compartments. In DC2.4 cells, the monensin enhancement for Man-tri (4b) and Man-tri-spa
(5b) (even Tri-Man (1b)) was high at low concentration and then dropped to even the value of
1 meaning that fluorescein was in a neutral environment which could result from escape of
liposomes from acidic compartments. However, it was difficult to conclude about different
intracellular processing of liposomes internalized via MR or DC-SIGN because the cell types
were different. Altogether, the results indicated that Man-tri (4b) provided better uptake by
DC-SIGN positive cells than MR positive cells. Conversely, we observed better selectivity
and uptake of Tri-Man (1b) by MR positive cells. MR is a monomeric membrane-bound
protein allowing mannose binding to two (or more) separate but interacting carbohydrate
recognition domains (CRDs) within the mannose receptor [34-36]. The structure of the tri-
antenna α-mannopyranoside in Tri-Man (1) is more adapted to bind to separate CRDs of MR
than the tri-mannose structure of Man-tri (4). Conversely, the presence of the branching α(1,2)
mannose on Man-Tri (4b) increases their binding toward DC-SIGN as reported [37].
Comparatively, PMan4 (6) and PMan5 (7) did not provide benefit in terms of binding and
uptake of the liposomes.
2.4. Binding and uptake of liposomes by hMo-DCs
9

The same experiments were performed on hMo-DCs that expressed both MR and DC-SIGN.
The MFI on the surface of the cells incubated with Tri-Man (1b), Man-tri (4b), Man-tri-spa
(5b), PMan4 (6b), and PMan5 (7b) was saturable indicating a receptor mediated binding of
those liposomes (Figure 5A).
In contrast, the binding of Man (3b) increased linearly with the concentration and was not
saturable. The intracellular MFI of Man-tri-spa (5b) was higher than Man-tri (4b) and it was
much higher than Tri-Man (1b) (Figure 5B). The intracellular MFI with PMan4 (6b) was close
to that with Tri-Man (1b) but higher than with PMan5 (7b).
3. Conclusion
This work revealed that decorating DPPC/DOPE liposomes with a tri-antenna α-
mannopyranoside (Tri-Man) (1) is beneficial for targeting liposomes to MR while decoration
with a [Manα1-3(Manα1-6)Man] (Man-tri) (4) is beneficial for targeting DC-SIGN.
Compared to Tri-Man (1) and Man-tri (4), there was no great benefit to use other complex
mannose structures such as PMan4 (6) and PMan5 (7) in terms of binding and uptake
efficiency. In contrast to MR expressed in various kind of cells, DC-SIGN is expressed
preponderantly on DCs at mucosal sites, in skin and lymph nodes in humans. In skin, DCs
express DC-SIGN but not Langerhans cells nor macrophages. Overall, the results suggest that
antigen carried by liposomes prepared with Man-tri (4) would be more selective in vivo for
10

DCs thanks to better recognition by DC-SIGN. Evaluating the selectivity of the targeting,
immune responses and therapeutic efficacy induced upon administration of antigens pay
loaded liposomes decorated with Tri-Man (1) or Man-tri (4) would be of great importance.
However, a murine model expressing DC-SIGN is imperative. Recently, it was reported that
mDC-SIGN mice expressing CD209a/SIGNR5 display overlapping similarities between
hDC-SIGN and CD209a/SIGNR5 [38]. A humanized DC-SIGN murine model is also
available [39]. These models may be an interesting approach for these investigations after
determining whether the binding and uptake of liposomes with Man-tri (4) and Tri-Man (1)
with their DCs are identical to that reported here on hDC.
4. Experimental
All reagents were purchased from Sigma unless otherwise stated and used without further
purification. The tri-antenna of α-D-mannopyranoside diether lipid (Tri-Man) (1) was
synthetized as described [40]. 1-azido-β-D-glucopyranoside (Glc-azide) (2a) and 1-azido-α-
D-mannopyranoside (Man-azide) (3a) were purchased from Sigma.
4.1.Synthesis of 3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranosylazide (Man-tri azide) (4a)
The sugar was synthesized as described [29].
4.2. Synthesis of 5-azidopentanoic acid
The compound was synthetized as described [41, 42]. To a stirred solution of 5-bromovaleric-
acid (5 g; 27.61 mmol) in dry DMF (50 mL) was added sodium azide (2.15 g; 33.14 mmol;
11

1.2 eq). The mixture was stirred for 2 h at 80°C, and then diluted with EtOAc (50 mL). The
organic phase was washed with brine (2 x 100 mL), water (2 x 100 mL), dried over sodium
sulfate, filtered and concentrated. The crude product was purified by “manual” flash
1
chromatography (Cyclohexane/ EtOAc, 5/5) to give the compound (1.90 g; 48%). H NMR
(CDCl3, 300 MHz) δ(ppm) 11.11 (s, 1H, COOH); 3.33 (t, J = 6.6 Hz, 2H, CH2d); 2.43 (t, J =
7 Hz, 2H, CH2a); 1.85-1.55 (m, 4H, CH2b, CH2c). 13C NMR (CDCl3, 75 MHz) δppm
179.6 (COOH); 51.0 (C-d); 33.4 (C-a); 28.1(C-c); 21.8 (C-b) (Supplementary Figure 5a).
4.3. Synthesis of 2,4-di-O-benzoyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-
-mannopyranosylamido-5-azido-pentanamide
α
-
D
-mannopyranosyl)-
β
-
D
A solution of 2,4-di-O-benzoyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-α-
D
-mannopyranosyl)-α-
D
-
mannopyranosylazide [43] (1.5 g, 1.4 mmol) in methanol (30 mL) was hydrogenated in a
Hcube (Mode Full H2, 1 mL.min-1, 25 °C) on a Pd/C 10 % cartridge for 36 h. Methanol was
removed under reduced pressure to afford the crude β-amine (1.45 g, 99%) pure enough for
the next step. The crude β-amine (1g; 0.95 mmol) was dissolved in dry dichloromethane
(CH2Cl2) (40 mL) at room temperature and in this addition order 5-azidopentanoic acid (200
mg; 1.15 mmol) beforehand dissolved in dry CH2Cl2 (1 mL), HATU (726 mg; 1.91 mmol)
and DIPEA (332 µL; 1.94 mmol) were added. The mixture was stirred under argon
atmosphere for 36 h, then diluted with CH2Cl2 (10 mL), the organic phase washed with
satured NaHCO3 (2 x 10 mL) and water (2 x 10 mL), dried over sodium sulfate, filtered, and
12

concentrated. The crude product was purified by “automatic” flash chromatography
1
(H2O/MeCN, 70/30 to 0/100) to afford the compound (620 mg; 55%). H NMR CDCl3,
300 MHz) δ(ppm) 8.19-7.40 (m, 10H, 2 C6H5COO); 6.52 (d, J = 9 Hz, 1H, NHCO); 5.75 (t, J
= 9.9 Hz, 1H, H-4A); 5.70-5.62 (m, 2H, H-1A + 1H); 5.37 (dd, J = 3.4 Hz, J = 10Hz, 1H, H-
3C); 5.31 (dd, J = 1.7 Hz, J = 3.4 Hz, 1H, H-2B or H-2C); 5.23 (t, J = 10 Hz, 1H, H-4C); 5.09
(t, J = 9.6 Hz, 1H, H-4B); 5.00 (dd, J = 3.5 Hz, J = 9.7 Hz, 1H, H-3B); 4.90 (d, J = 1.8 Hz,
1H, H-1B); 4.87-4.81 (m, 2H, H-1C + 1 H); 4.32-3.66 (m, 9H, H-6A, H-6’A, H-6B, H-6’B,
H-6C, H-6’C + 3 H); 3.26 (t, J = 6.4 Hz, 2H, CH2d); 2.25 (t, J = 6.7 Hz, 2H, CH2a); 2.11,
2.06, 1.99, 1.97, 1.97, 1.80, 1.77 (7s, 24H, CH3COO);1.74-1.55 (m, 4H, CH2b, CH2c).
13C NMR (75 MHz, CDCl3) δppm 171.83, 170.64, 170.57, 169.99, 169.73, 169.69, 169.44,
168.84, 166.42, 164.85 (C6H5COO, CH3COO, NHCO); 133.74, 133.59, 130.15, 129.82,
128.8, 128.79, 128.61, 128.50 (C6H5COO); 99.24, 97.96 (C-1B, C-1C); 76.88 (C-1A); 76.57,
74.72, 71.94, 69.50, 69.46, 69.05, 68.48, 68.08 ([C-2, C-3, C-5]-ABC, C-4A); 66.30 (C-6A);
65.92 (C-4B, C-4C); 62.29 (C-6B, C-6C); 51.03 (C-d); 35.48 (C-a); 28.16 (C-c); 22.23 (C-b);
20.82, 20.67, 20.61, 20.55, 20.36, 20.26 (CH3COO) (Supplementary Figure 5b). ES-HRMS:
[M + Na]+ = 1195.3706 m/z calculated for C53H64N4NaO26; found 1195.3715.
4.4. Synthesis of 3,6-di-O-(
α
-
D
-mannopyranosyl)-β-D-mannopyranosylamido-5-azido-
pentanamide (Man-tri-spa azide) (5a)
13

To a solution of 2,4-di-O-benzoyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-β-
D
-mannopyranosylamido-5-azido-pentanamide (128.5 mg, 0.122 mmol) dissolved in MeOH
(3.5 mL) was added 1M sodium methoxide solution (3.68 mL, 3.68 mmol; 30 equiv.) The
mixture was stirred for 7 days at 30°C then neutralized with Amberlite IR120 H+ resin,
filtered, and concentrated. The crude product was purified by “automatic” flash
chromatography (H2O/MeCN, 95/5 to 0/100) to afford the compound 5a (47.7 mg, 69%
containing 9% of α anomer). 1H NMR (D2O, 300 MHz) δ(ppm) 5.21 (d, J = 1 Hz, 1 H, H-1A)
5.11 and 4,84 (2d, J = 1.7Hz, 2H, H-1B, H-1C); 4.11-3.55 (m, 18H, H-2A to C, H-3A to C,
H-4A to C, H-5A to C, 2 H-6A to C)); 3.31 (t, J = 6.6 Hz, 2H, CH2d); 2.34 (t, J = 7 Hz, 2H,
CH2a); 1.73-1.48 (m, 4H, CH2b, CH2c). 13C NMR (75 MHz,D2O) δppm 176.91 (NHCO);
102.13, 99.52 (C-1B, C-1C); 80.69, 77.78, 75.77, 73.41, 72.70, 70.57, 70.38, 70.04, 69.88,
69.80, 66.84, 66.73, 66.66, 65.43 (C-1A, [C-2, C-3, C-4, C-5]-ABC, C-6A); 61.06, 60.96 (C-
6B, C-6C); 50.75 (C-d); 34.92 (C-a); 27.43 (C-c); 22.29 (C-b) (Supplementary Figure 5c).
ES-HRMS: [M + Na]+ = 651.2337 m/z calculated for C23H40N4NaO16; found 651.2338.
4.5. Synthesis of 2-Amidoethyl 3-O-[1-(2-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl)-
1,2,3-triazol-4-yl]methyl- -mannopyranoside-5-azido-pentanamide (PMan4azide) (6a)
α-D
A solution of 2-Aminoethyl 3-O-[1-(2-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl)-
1,2,3-triazol-4-yl]methyl-α-D-mannopyranoside hydroformate [29] (84 mg, 0.124 mmol) and
DIPEA (22 µL, 0.129 mmol) in DMF (5 mL) was stirred for 2h at room temperature under
14

argon. A solution of 5-azidopentanoic acid (18 mg, 0.125 mmol), DIC (40µL, 0.253 mmol)
and HOBt (34 mg, 0.251mmol) in DMF (5 mL) was prepared 2 h before use. The two
solutions were mixed and stirred for 24 h at 60 °C. DMF was removed in vacuo, the crude
product passed through a sephadex column and then purified by HPLC to afford compound
1
6a (22 mg, 23%) as a white solid. H NMR (D2O, 400 MHz) δ(ppm) 8.12 (s, 1H, H-5
triazole); 6,28 (d, J = 1,8 Hz, H, H-1B); 5.06 (d, J = 1.3 Hz, 1H, H-1C); 4.80-4.63 (m, 4H, H-
1A, H-2B, OCH2CCHN3); 4.15 (dd, J = 2.5 Hz, J = 6.7 Hz, 1H, H-3B); 4.07-3.99 (m, 2H, H-
2A, H-2C); 3.81-3.25(m, 18H, H-3A, H-3C, H-4A to C, H-5A to C, 2 H-6A to C, CH27,
CH28 ); 3.24 (t, J = 4.8 Hz, 2H, CH2d); 2.20 (t, J = 5.55 Hz, 2H, CH2a); 1.62-1.45 (m, 4H,
CH2b, CH2c). 13C NMR (D2O, 100 MHz) δppm 176.59 (NHCO); 144.49 (C-4 triazole);
124.71 (C-5 triazole); 101.96 (C-1C); 99.50 (C-1A); 84.89 (C-1B); 78.66, 76.58, 76.17,
73.49, 72.71, 70.21, 69.83, 69.78, 66.61, 65.84, 65.48, 61.57, 60.91, 60.76, 60.35 ([C-2, C-3,
C-4, C-5, C-6]-ABC, C-8 and OCH2(CCHN3)); 50,65 (C-d); 38,80 (C-7); 35.20 (C-c); 27.42
(C-b); 22.69 (C-a) (Supplementary Figure 5d). ES-MS: [M + Na]+ = 776.3 m/z calculated for
C28H47N7NaO17; found 776.6.
4.6. Synthesis of 2-Amidoethyl 3,6-di-O-[1-(
-mannopyranoside-5-azido-pentanamide (PMan5 azide) (7a)
A solution of 2-Aminoethyl 3,6-di-O-[1-(α- -mannopyranosyl)-1,2,3-triazol-4-yl]methyl-α-
α-D-mannopyranosyl)-1,2,3-triazol-4-yl]methyl-
α-D
D
D
-mannopyranosidehydroformate [29] (150 mg, 0.211mmol) and DIPEA (36 µL, 0.211
15

mmol) in DMF (5 mL) was stirred for 45 min at room temperature under argon. A solution of
5-azidopentanoic acid (31 mg, 0.216 mmol), DIC (71µL, 0.455 mmol) and HOBt (54 mg,
0.399 mmol) in DMF (5 mL) was prepared 30 min before use. The two solutions were mixed
and stirred for 24 h at 60 °C. DMF was removed in vacuo, the crude product passed through a
sephadex column and then purified by HPLC to afford compound 7a (25 mg, 11%) as a white
solid. 1H NMR (D2O, 400 MHz) d(ppm) 8.12, 8.11 (2s, 2H, H-5 triazole); 6.03, 6.02 (2d, J =
1.8 Hz, H-1B, H-1C); 4.77-4.60 (m, 7H, H-1A, H-2B, H-2C, 2 x OCH2(CCHN3)); 4.06-4.01
(m, 3H, H-2A, H-3B, H-3C); 3.76-3.58 (m, 13H, H-3A, H-4A to C, H-5A, 2 H-6A to C,
CH28); 3.51-3.15 (H-5B, H5-C, CH27, CH2d); 2.16 (t, J = 5.4 Hz, 2H, CH2a); 1.60-1.40 (m,
4H, CH2b, CH2c). 13C NMR (D2O, 100 MHz) δppm 176.66 (NHCO); 144.63 and 144.31 (C-
4 triazole); 124.92 and 124.89 (C-5 triazole); 99.80 (C-1A); 86.88 (C-1B and C-1C); 78.72,
76.26, 71.60, 70.63, 69.19, 68.42, 68.40, 66.80, 66.65, 66.37, 66.17, 65.67, 63.48, 61.80,
60.59 ([C-2, C-3, C-4, C-5, C-6]-ABC, C-8 and OCH2(CCHN3)); 50,83 (C-d); 39.03 (C-7);
35.39 (C-c); 27.63 (C-b); 22.90 (C-a) (Supplementary Figure 5e). ES-MS: [M + Na]+ = 857.3
m/z calculated for C31H50N10NaO17; found 857.5.
4.7. Synthesis of Propargyl-PEG8-amine
Azide-PEG8-alcohol (138 mg, 0.35 mmol) (Broadpharm) was solved in 5 ml DMF and cooled
to 0°C. NaH (17 mg, 0.7 mmol) was added portion wise and stirred for 10 min at 0 °C before
adding of 80% propargyl bromide solution (75 µl, 0.7 mmol). After stirring overnight at room
16

temperature, 20 ml water was added and the product was extracted with 2 x 25 ml ether. The
organic layer was dried on MgSO4, filtered and concentrated under pressure. The crude
product was purified by silica gel chromatography (CH2Cl2/CH3OH, 95:5) to yield azide
product as colorless oil (140 mg, 92%). The azide group was then reduced in amino group in
THF/water (4:1; v:v) in the presence of triphenylphosphine (109 mg, 0.45 mmol) under N2
atmosphere. The alkyne-PEG8-amine was used in next step without further purification.
4.8. Synthesis of Propargyl-PEG8-diether
The acid diether lipid (76 mg, 0.124 mmol) prepared as described [40] in 20 ml CH2Cl2
containing HBTU (52 mg, 0.136 mmol) was stirred at room temperature for 30 min. Then, the
propargyl-PEG8-Amine (46 mg, 0.113 mmol) and DIPEA (18 mg, 0.136 mmol) were added to
the above solution and stirred overnight at room temperature. The solution was washed with
aqueous solution of 1N HCl and brine. The organic phase was dried on MgSO4 and
concentrated under reduced pressure. The crude product purified by flash chromatography on
silica gel (CH2Cl2/CH3OH, 98/2) yielded alkyned-lipid as colorless oil (92 mg, 80%); Rf =
0.23 (DCM/CH3OH, 95/5). 1H NMR (CDCl3, 600 MHz) δ(ppm) 7.11-7.13 (m, 1H, NH); 4.08
(s, 2H, CH2C≡CH); 3.89-3.92 (m, 1H, CHC=O); 3.75-3.79 (m, 1H, CH2-O); 3.54-3.72 (m,
33H, CH2-O); 3.42-3.50 (m, 4H, CH2-NH,CH2-O); 3.32 (s, 1H, CH≡C); 1.06-1.81 (m, 52H,
24CH2, 4 CH); 0.84-0.87 (m,18H, 6 CH3).
17

4.9. Synthesis of Glycosylated diether Lipids 2-7
Propargyl-PEG8-diether (10 mg, 0.01 mmol) in 1 ml THF was added into 1 ml water
containing glycosyl-azide (0.012 mmol), CuSO4 (6 mM) and sodium ascorbate (6 mM). The
mixture was stirred overnight at room temperature. The glycolipids were purified by silica gel
chromatography (CH2Cl2/CH3OH/H2O, 90/10/1). Yield all>95%. Mass spectra : Glc (2) :
found 1227.71; theoretical 1227.89. Man (3) : found 1227.75; theoretical 1227.89. Man-tri
(4) : found 1552.11; theoretical 1051.99. Man-tri-spa (5) : found 1651.18; theoretical
1651.06. PMan4 (6) : found 1776.36; theoretical 1776.12. PMan5 (7) : found 1857.17;
theoretical 1857.16 (Supplementary Figures 6).
4.10. Liposomes
Liposomes 1b-7b were prepared at 5.4 mM in RNAse-free 10 mM Hepes buffer, pH 7.4
(Hepes). Lipids in ethanol were combined in
a
round bottom flask in
a
DPPC/DOPE/glycodiether-lipid (1-7)/Flu-DOPE ratio of 47.25%:47.25%:5%:0.5%. After
removal of the solvent by a rotary vacuum pump, the lipid film was dried for another 30 min
in vacuum, and then hydrated with Hepes. The liposomes were then subjected to five cycles
of freeze-thaw between Liquid N2 and 45 °C water bath for 2 min in each step. The size of the
liposomes was reduced by extruding 15 times at a mini extruder (Avanti Polar Lipid) through
polycarbonate membranes with a final pore size of 200 nm (Nuclepore).
4.11. Size and
ζ potential measurements
18

The size and ζ potential of liposomes were measured in Hepes by using the SZ-100 Analyser
(Horiba Scientific).
4.12. Man-Liposomes recognition by lectins
ConA from Canavaliaensiformis (Jack bean) binds to terminal α-D-mannosyl and α-D-
glucosyl residues with a Kd of 8.75 10-4 M for MeαMan. BC2LA a lectin from
Burkholderiacenocepacia displays mannose specificity with a Kd for MeαMan of 2.75 10-6 M
[44, 45]. It binds also to all αMan-terminating oligosaccharides. Interactions between
liposomes and lectins were measured by GLYcoPROFILE® technology from GLYcoDiag
(Orléans, France). This method is a lectin array-based assay which uses customized lectin
immobilized 96-well plate (LEctPROFILEpalte) (GLYcoDiag) [46]. Samples of liposomes
(2.7 mM) were diluted in PBS supplemented with 1mM CaCl2 and 0.5 mM MgCl2 and
incubated (50 µL / well) in lectin plates during 1h. After washing with PBS (three times),
fluorescence was read with the Fluostar (BMG labtech).
4.13. Cells and cell culture
All reagents were purchased from ThermoFisher Scientific unless otherwise stated and used
without further purification. The DC2.4 cell line was a murine dendritic cell line maintained
in RPMI supplemented with 10% fetal calf serum (FCS), glutamine (2 mM), penicillin (100
U/mL) and streptomycin (100 µg/mL) (complete medium) [47]. The DC-SIGN HEK293 cell
line – kindly given by O. Schwartz (Pasteur Institute, Paris, France) - was a human cell line
19

obtained by transduction of HEK293T cells with lentivirus encoding the DC-SIGN human
lectin (CD209) [30]. DC-SIGN HEK293 cells grew in DMEM complete medium. All cells
grew in humidified air with 5% CO2 at 37 °C and were Mycoplasma free when checked by
using the MycoAlert1 Mycoplasma Detection Kit (Lonza). hMo-DCs were obtained as
described [48]. PBMC were freshly isolated from leukapheresis residues (EFS, Paris, France)
collected from healthy volunteers after informed written consent). CD14-positive cells were
isolated from PBMCs using a magnetic cell sorting (MiltenyiBiotec) and then cultured in the
presence of 10% human AB serum RPMI medium, GM-CSF (100 ng/mL; Gentaur) and IL-4
(10 ng/mL; MiltenyiBiotec).
4.14. Binding and uptake of liposomes
Two days before experiments, DC2.4 and DC-SIGN expressing HEK293 cells were seeded in
24-well culture plates (1 × 105 cells/cm2). hMo-DCs were used after for 5 days of culture in
the presence of 10% human AB serum RPMI medium, GM-CSF and IL-4. The day of the
experiments, the medium was removed, the cells washed with PBS and incubated for 3h with
Flu-Lip in serum free medium. Cells were harvested without trypsin, collected into FACS
tubes and washed with PBS. The cell-associated fluorescence intensity was measured with a
flow cytometer (FORTESSA x 20, Becton Dickinson) with λex = 488 nm; λem = 530 ± 30
nm. The fluorescence intensity was expressed as the mean fluorescence intensity (MFI) of 10
000 cells. Each tube was analyzed three times. The first time in PBS (total fluorescence), the
20

second time in the presence of Trypan blue (TB) added into the tube at a final concentration
of 0.004 % to quench the fluorescein fluorescence associated on the surface of cells. The third
time, the fluorescence intensity was measured in PBS in the presence of monensin added 30
min before measurement at a final concentration of 0.03 mg/mL in order to measure the
fluorescence intensity upon neutralization of acid intracellular compartments [26, 49].
Acknowledgements
We warmly thank Dr. L. Landemarre from GLYcoDiag (Orléans, France). We thank D.
Gosset1, the P@CYFIC platform at CBM and C. Baillou3 for their technical assistance in flow
cytometry. The authors acknowledge Drs. D. Lesur (UMR 7378) and S. Pilard (Plateforme
Analytique, UPJV, France) for MS and HPLC. This work was supported by grants from
“Région Centre Val de Loire” (APR IR 2015 VACARME project), European Community
(Univax:www.UniVax-FP7.eu). Haifei Gao received 2 years fellowship from “Région Centre
Val de Loire” (APR IR 2015 VACARME project). We certify that there is no conflict of
interest, no competing interest and no disclosure.
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Figure Legends
Figure 1: (A) Structure of the trimannosylated diether lipid (Tri-Man) (1). (B) Glycal azide
structures. 1-azido-β-D-glucopyranoside (Glc-azide) (2a); 1-azido-α-D-mannopyranoside
(Man-azide) (3a); 1-azido-3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside (Man-tri-
azide) (4a); 1-N-butylazide, 3,6-di-O-(α-D-mannopyranosyl)-β-D-mannopyranoside (Man-
tri-spa-azide) (5a); 1-ethyl-amino-butylazide Pseudo-Man4 (PMan4-azide) (6a); 1-ethyl-
amino-butylazide Pseudo-Man5 (PMan5-azide) (7a).
Figure 2: Synthesis of the Propargyl-PEG8-diether and glycal-diether lipids
Figure 3: Binding of Mannosylated Liposomes to mannose specific lectins. Flu-liposomes
were incubated with (A) ConA or (B) BC2LA lectins linked on plastic. After washing the
fluorescence intensity was measured at 520 nm upon excitation at 495 nm. Liposomes (○) Tri-
26

Man (1b); (♦) Glc (2b); (□) Man (3b); (■) Man-tri (4b); (●) Man-tri-spa (5b); (▲) PMan4
(6b); (∆) PMan5 (7b).
Figure 4: Binding and uptake of mannosylated liposomes by DC2.4 cells and DC-SIGN
HEK293 cells. The fluorescence intensity of cells incubated with Flu-liposomes was
measured by flow cytometry and analysis as described in Supplementary Figure 4. Binding of
liposomes on DC2.4 (Aa) and DC-SIGN HEK293 (Ab) cells. Intracellular quantity of
liposomes in (Ba) DC2.4 and (Bb) DC-SIGN HEK293 cells. Monensin enhancement in
DC2.4 (Ca) and DC-SIGN HEK293 (Cb) cells. Liposomes (○) Tri-Man (1b); (♦) Glc (2b);
(□) Man (3b); (■) Man-tri (4b); (●) Man-tri-spa (5b); (▲) PMan4 (6b); (∆) PMan5 (7b). MFI
of each sample was measured three times.
Figure 5: Binding and uptake of mannosylated liposomes by hMo-DCs. The fluorescence
intensity of cells incubated with Flu-liposomes was measured by flow cytometry and analysis
as described in Supplementary Figure 4. (A) Binding of liposomes (○) Tri-Man (1b); (□) Man
(3b); (■) Man-tri (4b); (●) Man-tri-spa (5b); (▲) PMan4 (6b); (∆) PMan5 (7b). (B)
intracellular quantity of liposomes (○) Tri-Man (1b); (□) Man (3b); (■) Man-tri (4b); (●)
Man-tri-spa (5b) or PMan4 (6b) or Glc (2b); (∆) PMan5 (7b). MFI of each sample was
measured three times. MFI of each sample was measured three times.
27

28