Targeting Human Dendritic Cells with Lewis X Modified Liposomes

In this short report, we demonstrate that liposomes bearing the Lewis X trisaccharide on the surface (“Awesosomes”) efficiently target human dendritic cells. We chose a glycolipid with Lewis X trisaccharide headgroup to facilitate the targeted liposome uptake via the DC-SIGN internalization pathway. While no uptake of Awesosomes was detected with wild-type human HEK293 cells, HEK293 cells transfected with human DC-SIGN internalized Awesosomes extensively. In samples of human blood-derived leukocytes, the extent of uptake of Awesosomes correlated with the expression of DCSIGN, which is a dendritic cells marker. There was a marked difference in the uptake of Awesosomes and plain liposomes by DC-SIGN expressing dendritic cells. There was no difference in uptake of Awesosomes and plain liposomes by wt HEK293 cells or macrophages. These results indicate that Lewis X trisaccharide can “sweet-talk” dendritic cells into internalizing a delivery vehicle, and that Awesosomes are promising as “magic bullets” for specific delivery of drugs, antigens, or immunostimulatory molecules to human dendritic cells without influencing other cell types. Introduction Dendritic cells (DCs) are the most powerful antigen presenting cells. They are extremely potent in priming naïve T-cells (and have been therefore designated the “nature’s adjuvants” [Steinman, 2007], and in inducing cytotoxic immune responses (CTL). Cytotoxic response is required for an efficient protection against a number of pathogens that evade immune response, and also against cancer. Importantly, DCs are capable of crosspresentation, the induction of a cytotoxic cell response against exogenously acquired antigen [Bevan, 1976]. This mode of CTLs induction against an exogenous antigen has been described only relatively recently and is extremely important for developing vaccines against certain pathogens including malaria, HIV, and TB [Winau, 2006], but also against tumor cells. In addition to antigen targeting to DCs, which has already been described [Bonifaz, 2004; Sancho, 2009] and shown to increase vaccine performance, we believe that also targeting of immunostimulatory molecules and other adjuvants may be beneficial. Targeted delivery of immunostimulants should decrease the likelihood of mounting a generalized inflammatory response; current adjuvants elicit a generalized inflammatory response, often leading to a number of side effects like swelling. Thus, our aim was to find a way to target a universal delivery vehicle suitable for delivery of both antigens and adjuvants to DCs. Liposomes are an ideal vehicle because they provide the interior aqueous environment for encapsulation of hydrophilic molecules (e.g. protein and peptide antigens, or soluble immunostimulatory molecules like CpG or poly I:C), and the lipid membrane, which can harbor hydrophobic molecules (acyl-modified peptides, TLR4 agonists derived from lipid A, etc.). We chose to target DC-SIGN receptor, a C-type lectin for which the ligand profile has been thoroughly characterized and high-affinity glycan ligands have been identified [Guo, 2004]. Lewis X trisaccharide has been identified as one of the best DC-SIGN ligands. Esnault et al. described the synthesis of highly hydrophic Lewis X glycolipids [Esnault, 2001] which bind DC-SIGN. We show that POPC liposomes with 0.5mol% of this Lewis X compound are efficiently taken up by human blood derived dendritic cells. Materials and Methods Preparation of plain liposomes and Awesosomes Lipids were mixed in chloroform and dried under a stream of nitrogen. The resulting film was hydrated with 20mM N at ur e P re ce di ng s : d oi :1 0. 10 38 /n pr e. 20 10 .4 76 4. 1 : P os te d 12 A ug 2 01 0 HEPES/130mM NaCl, pH 7.25, vortexed, sonicated, and allowed to go through 1 to 5 freeze-thaw cycles before extrusion through 100nm polycarbonate membrane at a temperature equal or higher than that of the Tm of the lipids. Transfection of HEK293 cells with DC-SIGN HEK293 cells were seeded at a density of ~25000 cells / cm. Next day, they were transfected using the FuGENE (Roche) transfection reagent as follows: 1.5g pcDNA3.1 containing the human DC-SIGN (a kind gift of Benhur Lee, LA) was mixed with 4.5l FuGENE in 100ul serum-free MEM media, and allowed to complex for 10-30’. The mixture was then added to the cells in complete media (0.1g DNA was enough for 1cm cells). The transfected cells were then incubated O/N to allow for the expression of DC-SIGN. Cell imaging and immunofluorescence In order to visualize the uptake of liposomes, 0.05% of DiO-C18 or DiI-C18 (Invitrogen) was included in the lipid mix. For viewing, the cells were washed extensively with PBS/0.2% BSA/0.5% gelatin/2mM EDTA, fixed with 4% PFA (DAPI was included when required), and when appropriate, blocked with PBS/gelatin and stained with FITC-conjugated anti-DC-SIGN antibody (R&D FAB161F, used at a recommended dilution). The cells were then washed again and finally mounted in mowiol for viewing. Liposome internalization assays Cells were grown in 96 well plates to confluency. Liposomes were added at a concentration of 0.3mM lipid, mixed, and incubated as indicated. When the incubation period was over, the cells were chilled quickly on ice, washed extensively with PBS/gelatin/EDTA, and the fluorescence was measured in PBS. New synthetic method of Lewis X neolipid The Lewis neolipid 1 used in this study was originally synthesized by our group for study of its monolayer behaviour [Esnault, 2001]. A more efficient synthetic route for this compound has been developed recently in our group which was depicted in Scheme 1. The known pentasaccharide trichloroacetimidate 2, readily obtained by our previous report [Gourier, 2005], was condensed with the known alcohol 3 [Esnault, 2001]. The reaction was promoted by trimethylsilyl triflate (TMSOTf) in dichloromethane in the presence of molecular sieve (4Å) at 0°C for 4h to give the protected Lewis neolipid 4 in 68% yield. De-O-acetylation of compound 4 by sodium methoxide in methanol and dichloromethane at room temperature for 3h provided the target product 1 in 98% yield. Both compounds 1 and 4 were fully characterized by H NMR, C NMR and Mass Spectroscopy analyses.