BODIPY-Labeled DC-SIGN-Targeting ... - ACS Publications

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BODIPY-Labeled DC-SIGN-Targeting Glycodendrons Efficiently Internalize and Route to Lysosomes in Human Dendritic Cells Renato Ribeiro-Viana,†,# Juan J. García-Vallejo,‡,# Daniel Collado,§,∥ Ezequiel Pérez-Inestrosa,§,∥ Karien Bloem,‡ Yvette van Kooyk,‡ and Javier Rojo*,† †

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC − Universidad de Sevilla, Avenida Américo Vespucio 49, Seville 41092 Spain ‡ Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands § Andalusian Centre of Nanomedicine and Biotechnology-BIONAND, Parque Tecnológico de Andalucía, 29590 Malaga, Spain ∥ Department of Organic Chemistry, Faculty of Science, University of Malaga, 29071 Malaga, Spain S Supporting Information *

ABSTRACT: Glycodendrons bearing nine copies of mannoses or fucoses have been prepared by an efficient convergent strategy based on Cu(I) catalyzed azide−alkyne cycloaddition (CuAAC). These glycodendrons present a well-defined structure and have an adequate size and shape to interact efficiently with the C-type lectin DCSIGN. We have selected a BODIPY derivative to label these glycodendrons due to its interesting physical and chemical properties as chromophore. These BODIPY-labeled glycodendrons were internalized into dendritic cells by mean of DC-SIGN. The internalized mannosylated and fucosylated dendrons are colocalized with LAMP1, which suggests routing to lysosomes. The interaction of these glycodendrons with DC-SIGN at the surface of dendritic cells did not induce maturation of the cells. Signaling analysis by checking different cytokines indicated also the lack of induction the expression of inflammatory and noninflamatory cytokines by these second generation glycodendrons.

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INTRODUCTION New synthetic strategies should be addressed to design novel immunotherapeutic approaches in diseases where existing vaccines are inefficient (cancer, HIV, etc.).1 These elusive cases have led to the exploration of different approaches in order to find the adequate manner to initiate a protective adaptive immune response.2−11 Mannosylation of antigens (peptides or proteins) can be considered a very interesting strategy to modulate and increase adaptive immune responses. In 1997, Koning12 and Engering13 demonstrated simultaneously that mannosylation of a protein resulted in a 100-fold increase in the uptake of these antigens by dendritic cells (DCs). The uptake was mediated by mannose-binding receptors and led to proper antigen processing and presentation to T cells. Since these initial publications, other examples using a strategy based on the direct mannosylation of the antigen have been reported in the literature.14−16 Although some positive results were reported, the above-mentioned strategy presents an important drawback: direct mannosylation could interfere negatively in the final biological effect due to the masking of important epitopes, the modification of antigen conformation, the appearance of new but irrelevant epitopes, and so on. Moreover, the lack of control during the mannosylation reaction leads to the generation of random mannosylated antigens. In other words, direct mannosylation of antigens results in poorly characterized polydisperse products. © XXXX American Chemical Society

A different strategy extensively used is based on the generation of a peptide antigen constructed using a solid phase peptide synthesis (SPPS) approach containing a multiple antigen peptide (MAP) or a polylysine peptide to be mannosylated on the amino groups of the side chains.17−20 This strategy allows a control of the final structure but is limited by the small number of mannose ligands introduced in a final step. Nanoparticles have been also used as interesting platforms for a multivalent presentation of mannosides and antigens, although they present a polydispersity and use a relative large number of ligands than probably are not really required.21,22 The group of Pietersz et al.23,24 have proposed the use of poly(amidoamine) (PAMAM) mannosylated dendrimers conjugated to a model antigen, the ovalbumin protein (OVA). The use of mannosylated dendrimers provides a multimeric platform for efficient targeting, reducing the number of modified residues per molecule of antigen. Yet, this synthetic approach still results in the production of a polydisperse product where one or two OVA molecules were conjugated to the mannosylated PAMAM dendrimer. Several glycan binding proteins exist with a preferred affinity for mannose on the surface of antigen-presenting cells. All these Received: June 29, 2012 Revised: August 23, 2012

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dx.doi.org/10.1021/bm300998c | Biomacromolecules XXXX, XXX, XXX−XXX

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spectra are expressed in parts per million (ppm) relative to the residual solvent signal using manufacturer indirect referencing method. Signals were abbreviated as s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. Mass spectra were obtained with a Bruker ion-trap Esquire 6000 apparatus (ESI) and a Bruker ULTRAFLEX III (MALDI-ToF) from Bruker Daltonics; high-resolution mass spectrometry (HRMS) spectra were obtained with an Apex II instrument (FT-ICR, ESI) and with a Micromass Autospec-Q (FAB). Synthesis. Compounds 1 and 2,51 3a,52 3b,53 4a, 5a, 6, 7a, and 8a54 have previously been described. For the synthetic details and characterization of compounds 3c, 4b−c, 5b−c, 7b−c, 8b−c, 9, 10,55 11, and 12a−c, see the Supporting Information. General Method for Click Chemistry Reactions (4b−c, 7b−c, 12a−c). The azido derivative (3b−c, 5b−c, or 8a−c), the alkynyl compound (2, 6, or 11), a copper salt (CuSO4·5H2O or CuBr), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and sodium ascorbate (when Cu(II) is used as copper salt) were dissolved in THF/H2O (1:1) or CH3CN/H2O (1:1) in a sealed microwave vial. The solution was heated at 60 °C in a microwave oven for 20−24 min. A copper scavenger resin, QuadrasilMP, was added to the reaction solution and stirred for 5 min. After that, the mixture was filtered, and the resulting solution was purified by size-exclusion chromatography. General Method for Substitution of Chloro by an Azido Group (3c, 5b−c, 8b−c). Sodium azide (large excess) and the corresponding compound (4b−c, 7b−c, or 9) were dissolved in dimethylformamide (DMF). The mixture was stirred at 60 °C for 3 days. After that time, the solvent was concentrated, and the crude was purified.

receptors belong to the C-type lectin receptor (CLR) family and have an EPN motif in the carbohydrate recognition domain.25 Among the CLRs involved in mannose recognition, the mannose receptor (MR) and DC-SIGN have been proposed to be the most important ones. DC-SIGN has attracted a large interest since 2000,26 due to its key role in the trans-infection of T-cells by HIV,27 but also as a receptor for a multitude of pathogens.28 DC-SIGN is expressed mainly on the surface of DCs, and is able to recognize mannoses and fucoses on highly glycosylated proteins on the surface of pathogens. The cytoplasmic domain of DC-SIGN carries motifs involved in both internalization and signaling, two necessary events for the efficient capture, processing, and presentation of pathogenic antigens to T cells. Moreover, the signaling plays a crucial role in the costimulation and priming of the T cells.29−32 Thus the biological properties of DC-SIGN make this receptor an ideal candidate for the targeting of antigens to DCs with the final goal of modulating the immune response. Our experience in the synthesis of multivalent carbohydrate systems prompted us to design multivalent systems bifunctionalized with carbohydrates (mannoses or fucoses) and other molecules of interest to trigger both internalization and signaling into DCs via DC-SIGN. With the aim of designing multivalent monodisperse mannosylated and fucosylated compounds, we decided to address the preparation of simple mannosylated and fucosylated compounds with full control of the structure. Our approach was based on the extensive use of the click chemistry reaction based on the Cu(I) catalyzed azide−alkyne cycloaddition (CuAAC)33−35 The click chemistry reaction and in particular, the CuAAC, is a very efficient, versatile, and popular reaction that has found many applications in different fields to achieve the preparation of very diverse molecules ranging from small compounds to polymers, hydrogels, and other large entities. Also, this reaction has been used to conjugate biomolecules (proteins, DNA, etc.) under very mild conditions. Hundreds of papers have been published covering these applications, and some general reviews have also been published on this topic.36−43 This CuAAC reaction has been used previously to very efficiently prepare different glycodendrimers.44−50 We have used the CuAAC reaction to obtain glycodendrons in a convergent approach. These glycodendrons present at the focal position an appropriate functional group to allow the conjugation to other biomolecules of interest (peptides, nucleic acids, etc.), again using CuAAC. In order to be able to track the compounds, we have coupled a BODIPY derivative to the glycodendrons. This approach will allow us to follow the internalization and endocytic routing of DC-SIGN-targeting glycodendrons in DCs and evaluate their influence in antigen processing and presentation.

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DC-SIGN ELISA Glycodendrons were coated in phosphate-buffered saline (PBS) at indicated concentrations on NUNC maxisorb plates (Roskilde, Denmark) overnight at room temperature. Plates were blocked with 1% bovine serum albumin (BSA), and DCSIGN-Fc was added (0.5 μg/mL) for 2 h at room temperature in the presence or absence of 10 mM ethylene glycol tetraacetic acid (EGTA), 100 mM mannan, or 20 μg/mL AZN-D1. Binding was detected using a peroxidase-labeled antihuman IgG-Fc antibody (Jackson, West groove, PA). DC-SIGN-Fc, comprising the extracellular domains of DC-SIGN fused to the human IgG1 Fc tail, was generated as previously described.56 Fc-proteins were purified from the supernatant of CHOtransfectants using protA columns. Cells. Human Monocyte-Derived DCs. Monocytes were isolated from the blood of healthy donors (Sanquin, Amsterdam, Netherlands) through a sequential Ficoll/Percoll gradient centrifugation. Isolated monocytes (purity >85%) were cultured in RPMI 1640 (Invitrogen, Gibco, CA) supplemented with 10% fetal calf serum (FCS) (BioWhittaker, Walkersville, MD), 1,000 U/ml penicillin (BioWhittaker, Walkersville, MD), 1,000 U/ml streptomycin (BioWhittaker, Walkersville, MD), and 2 mM glutamine (BioWhittaker, Walkersville, MD) in the presence of interleukin-4 (IL-4) (500 U/ml; Biosource, CA) and granulocyte-macrophage colony-stimulating factor (GMCSF) (800 U/ml; Biosource, CA) for 7 days.57 DC differentiation was confirmed by flow cytometric analysis (FACScan, BD biosciences) of the expression of DC-SIGN using the monoclonal antibody AZN-D126 followed by staining with a secondary fluorescein isothiocyanate (FITC)-labeled antimouse antibody (Zymed, San Francisco, CA). K562/DC-SIGN and K562/DC-SIGNLL/AA. Stable K562/DCSIGN and K562/DC-SIGNLL/AA transfectants58 were maintained in RPMI 1640 medium containing 10% FCS, 1000 U/ mL penicillin (BioWhittaker, Walkersville, MD), 1000 U/mL streptomycin (BioWhittaker, Walkersville, MD), and 2 mM glutamine (BioWhittaker, Walkersville, MD). K562/DC-

MATERIALS AND METHODS

Reagents were purchased from Sigma-Aldrich, Senn Chemicals, and Flucka, and were used without purification. Solvents were dried by standard procedures. Reactions requiring anhydrous conditions were performed under argon. Synthetic compounds were purified by flash chromatography (FC) using medium or fine silica gel or by sephadex (LH20, G25). Thin layer chromatography (TLC) was carried out with precoated Merck F254 silica gel plates. FC was carried out with Macherey-Nagel silica gel 60 (230−400 mesh). Reaction completion was observed by TLC using as development reagents phosphomolibdic acid, 10% sulfuric acid in methanol or anisaldehyde. 1H, and 13C spectra were recorded on Bruker Avance DPX 300, DRX 400, and DRX 500 MHz spectrometers. Chemical shifts (δ) for 1H and 13C B


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from single stained samples to allow for compensation. Compensation samples were acquired with all channels habilitated and with the brightfield illumination and the 785 nm laser switched off. A minimum of 5000 cells from the single stained samples were acquired with the same settings as experimental samples to control for over/under compensation. Analysis was performed using the IDEAS v5.0 software (Amnis corp., Seattle). A compensation table was generated using the compensation macro built in the software. Single stained samples were manually gated for accurate calculation of spectral overlap coefficients.60 Once the compensation table was calculated, it was applied to the single staining samples that were acquired using the same settings as experimental samples. Proper compensation was then verified by visualizing samples in bivariate fluorescence intensity plots (data not shown). Then, a template analysis file was generated that included an area versus aspect ratio intensity plot and a gradient root-meansquare (rms) histogram of one of the brightfield channels (Channels 1 and 9). Area is the number of squared micrometers of the cells, while the aspect ratio intensity index is the result of dividing the minor axis (intensityweighted) by the major axis (intensity weighted) and describes how round or oblong an object is, but also indicates whether there are doublets in a population of normally circular cells. The gradient rms feature measures the sharpness quality of an image by detecting large changes of pixel values in the image and is useful for the selection of focused objects. The gradient rms feature is computed using the average gradient of a pixel normalized for variations in intensity levels. Using these features, a population of focused single cells (SC/F) was gated. This template, together with the corresponding compensation table was applied to all the experimental samples acquired. Each of the data files generated was opened, and the SC/F population was gated to a new compensated image file. Compensated image files were then merged into the final analysis file. This file allows for the direct comparison of features among the different glycodendrons. To calculate the glycodendron internalization, a mask was designed that characterizes only the intracellular space of the cells. This mask was based on the use of the morphology feature applied to the brightfield image on channel 1, and then eroded until the membrane was left out of the mask. Since cells are gated on a certain level of focusing, it is possible to assume that the image acquired represents, in all cells, a 4 μm crosssection of the major circumference.61 At this location, the thickness of the membrane is similar in all cells and allows us to design a mask based exclusively on brightfield images. This is a major advantage over the use of an extracellular fluorescent marker, which introduces new challenges in the experiment: the use of an additional channel complicates the compensation process, and the selection of a marker that is exclusively located in the extracellular membrane during the internalization process of the glycodendrons is a difficult task. The intracellular mask was then used to calculate the feature internalization applied to channel 2 (BODIPY). The internalization score is a log-scaled ratio of the intensity inside the cell (intracellular mask) with respect to the intensity of the entire cell. Cells that have internalized antigen typically have positive scores, while cells that show the antigen still on the membrane have negative scores. Cells with scores around 0 have similar amounts of antigen on the membrane and in intracellular compartments. Colocalization is calculated as the logarithmic transformation of Pearson’s correlation coefficient of the localized bright spots

SIGNLL/AA cells carry a mutation in the intracellular domain that DC-SIGN inhibits internalization of the receptor, while K562/DC-SIGN cells carry the wild-type complementary DNA (cDNA) for DC-SIGN and are able to mediate internalization. DC-SIGN expression was regularly selected using 1 mg/mL Geneticin (Invitrogen). To check for DC-SIGN expression, cells were incubated with primary antibody (AZN-D1)26 followed by staining with a secondary FITC-labeled antimouse antibody (Zymed, San Francisco, CA) and analyzed by flow cytometry on a FACScan (BD Biosciences, San Diego, CA). Maturation Assay. DCs were tested for maturation by flow cytometric analysis (FACScan, BD Biosciences) of the markers CD83 (PE-labeled, Beckman Coulter, Fullerton, CA), CD80, and CD86 (PE-labeled, both from BD Biosciences). Cytokine ELISA. For the detection of cytokines, culture supernatants were harvested 24 h after DC activation and frozen at −80 °C until analysis. Cytokines were measured by enzyme-linked immunosorbant assay (ELISA) using antibody pairs for human IL-6, IL-10, TNFα, IL-12p40, and IL-12p70 according to manufacturer (eBioscience). Real-Time PCR. Cells were lysed, and mRNA was isolated using an mRNA Capture kit (Roche, Basel, Switzerland). cDNA was synthesized using the Reverse Transcription System kit (Promega, WI) according to the manufacturer’s guidelines. Oligonucleotides were designed by using the computer software Primer Express 2.0 (Applied Biosystems) and synthesized by Invitrogen Life Technologies (Invitrogen, Breda, Netherlands). Real-time PCR analysis was performed as previously described with the SYBR green method in an ABI 7900HT sequence detection system (Applied Biosystems), using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous reference.59 Cellular Binding Assay. K562/DC-SIGNLL/AA cells were incubated in the presence of a range of concentrations of glycodendrons in the presence or absence of 10 mM EGTA, 100 mM mannan, or 20 μg/mL AZN-D1. Incubation was performed at room temperature for 60 min. Cells were then washed, and fluorescence was measured by flow cytometry (FACScan, BD Biosciences). Internalization and Intracellular Routing Assay. K562/ DC-SIGN cells were incubated in the presence of glycodendrons at either 4 or 37 °C for 60 min. Cells were then washed, fixed, and prepared for acquisition on the ImageStreamX (Amnis corp., Seattle) imaging flow cytometer. For the intracellular routing assay, DCs were incubated in the presence of glycodendrons at either 4 or 37 °C for 60 min. Cells were then washed, fixed, permeabilized, and stained with monoclonal antibodies against the early endosomal marker EEA1 and the lysosomal marker LAMP1. An AF594-labeled goat antimouse antibody was used as a secondary staining. The following laser powers were used for the internalization assay: 488 nm at 20 mW and 785 nm at 4.5 mW. Brightfield illumination was set at 800 mW before the acquisition of each sample. Brightfield images were collected in channels 1 and 9. Channels 2 (BODIPY) and 6 (granularity) were habilitated for the internalization assay, while channels 2 (BODIPY), 4 (AF594), and 6 (granularity) were habilitated for the intracellular routing assay. Cells were acquired at 40x magnification and on the basis of their area (area = the number of pixels in an image reported in square micrometers). Minimum area for acquisition was set to 50 pixels. A minimum of 15000 cells was acquired per sample at a flow rate ranging between 50 and 100 cells/s. At least 2000 cells were acquired C


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Scheme 1. Synthesis of Glycodendrons 8a−c Using CuAAC Reactions

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with a radius of 3 pixels or less within the whole cell area in the two input images (bright detail similarity R3). Since the bright spots in the two images are either correlated (in the same spatial location) or uncorrelated (in different spatial locations), the correlation coefficient varies between 0 (uncorrelated) and 1 (perfect correlation). The logarithmic transformation of the correlation coefficient allows the use of a wider range for the colocalization score. In general, cells with a low degree of colocalization or no colocalization at all between two probes show scores below 1. Since only molecules that have internalized are able to show colocalization with intracellular compartment markers, bivariate plots depicting the internalization score (Y axis) and the colocalization score (X axis) provide the best representation of data. In this scatter plot, a gate representing the cells that have internalized the probe and show colocalization was calculated, and the percentage of cells within that plot was calculated.

RESULTS

Our laboratory has developed a convergent strategy based on the Cu(I) catalyzed azide−alkyne cycloaddition (CuAAC), also known as click chemistry reaction, to allow an efficient synthesis of glycodendrons. In this approach, both the carbohydrate unit conjugation and the construction of the multivalent scaffolds were achieved using the CuAAC reaction. On the basis of this strategy, we have prepared glycodendrons of first and second generations conveniently functionalized at the focal position. The functional group at this position can be easily used to conjugate a fluorophore, which allows the tracking of the glycodendron in biological assays. A BODIPY derivative was chosen as fluorophore for its good photophysical and photochemical properties.62−64 The BODIPY derivative was conveniently functionalized with an alkyne group in order to allow the conjugation via a CuAAC reaction with an azido group at the focal position of the glycodendrons. D


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Scheme 2. Synthesis of the Maltotriose Derivative 3c

the second-generation glycodendron. This dendron 5a was conjugated with the trivalent scaffold 6 (similarly prepared as compound 2 but with a longer spacer) by a CuAAC reaction in the presence of CuSO4, sodium ascorbate, and TBTA. After substitution of the chloride by an azido group at the focal position using NaN3 in DMF, compound 8a was obtained in good yield. This nonavalent dendron 8a has been described previously by our group.54 We present here the extension of this strategy for the preparation of the other two glycodendrons with fucoses 8b and maltotrioses 8c. The fucosylated dendron was obtained using the same synthetic route as for mannose. The fucosyl derivative 3b was coupled to trivalent alkyne 2 using 0.2 equiv of CuBr and TBTA under microwave irradiation at 60 °C for 20 min to provide the trivalent fucosyl dendron 4b in 92% yield. After substitution of chloride by the azido group using sodium azide in DMF, the resulting dendron 5b was coupled to the trivalent alkyne 6 using CuSO4 as a copper source in the presence of sodium ascorbate and TBTA using again microwave irradiation for 20 min at 60 °C. Under these conditions, the glycodendron 7b was obtained in 75% yield. The compound was completely characterized by mass spectrometry (MS-ESI) and NMR. Finally, the substitution of chloride by azide afforded the fucosylated dendron 8b conveniently functionalized to be labeled with a chromophore. In the case of the maltotriose ligand, the CuAAC reaction to prepare the trivalent dendron 5c using deprotected maltotriose was not effective, probably due to the large number of free hydroxyl groups present, which can hijack the catalyst. The reaction evolved very slowly without the completion of all conjugations, complicating the isolation of a pure compound after chromatography. For this reason, the trivalent maltotriose 5c was prepared using the fully protected maltotriose 3c and CuSO4/sodium ascorbate/TBTA in THF/H2O at 60 °C using microwave irradiation. After the substitution of chloride by the azido group, compound 5c was conjugated with the trivalent scaffold 6 to afford the nonavalent dendron 7c in good yield. Finally, the conveniently functionalized glycodendron 8c was prepared and fully characterized by mass spectrometry and NMR. Glycodendrons 8 present the adequate functionality at the focal position to conjugate a fluorescence tag using a click chemistry reaction. A BODIPY derivative 11 bearing a primary alkyne group was prepared in order to incorporate the BODIPY chromophore into the dendrimeric structure via CuAAC reaction. To synthesize this chromophore 11, we first introduced the propargyl ether moiety (Scheme 3) by reacting 4-hydroxybenzaldehyde with propargyl bromide under basic conditions

A second-generation glycodendron bearing nine carbohydrates was selected due to its adequate size to interact efficiently with DC-SIGN. We have previously evaluated the binding process of several mannosylated dendrimers with this lectin, and we have demonstrated that mannosylated systems with a valency higher than 6 are appropriated to be recognized by DC-SIGN with a reasonable affinity (unpublished results). On the basis of our experience, we decided to address the synthesis of glycodendrons presenting nine copies of carbohydrates as depicted in Scheme 1. Mannose, fucose, and maltotriose have been selected as carbohydrate ligands to prepare the corresponding glycodendrons. All of these carbohydrates have a short linker at the anomeric position with a terminal azido group for the conjugation on the dendritic scaffolds via CuAAC reaction as described in Scheme 1. Mannose and fucose are monosaccharides recognized by DC-SIGN; however, maltotriose is not recognized by this lectin and will be used as a negative control in the interaction and internalization assays. To address the synthesis of glycodendrons, carbohydrates conveniently functionalized (3) were prepared. The synthesis of mannose 3a52 and fucose 3b53 has been previously described, and maltotriose 3c was synthesized as described in Scheme 2. Maltotriose was peracetylated using acetic anhydride and sodium acetate at 80 °C in quantitative yield as reported in the literature.65 The introduction of a short spacer, 2-bromoethanol, at the anomeric position was achieved with moderate yield using an excess of BF3·Et2O as promoter. The substitution of bromide by an azido group was performed by reaction with sodium azide at 80 °C in DMF in good yield. Finally, deprotection of the acetyl groups took place in quantitative yield using sodium methoxide in methanol. Once the carbohydrate derivatives 3 were prepared, we undertook the preparation of the multivalent scaffold. The commercially available pentaerythritol was selected as the core to synthesize glycodendrons 4. Three alkyne groups were introduced on the core as described in the bibliography using the fourth position to attach a short spacer by reaction with 2-chloroethyl ether.51 On this core, three carbohydrate units 3 conveniently functionalized with azido groups were conjugated using a CuAAC reaction to provide the key trivalent dendron 4. The mannosylated dendron 4a was previously prepared,54 but we have now improved this synthesis heating the reaction at 60 °C by the use of microwave irradiation during the cycloaddition step. Although yields in this reaction remain basically the same, the reaction times were notably reduced from many hours to minutes. The modification of the functional group at the focal position of this small dendron provides the building block 5a to create E


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similar way) were measured in saline−PBS aqueous media, a more related solvent to the cellular environment. Fluorescent properties of dendrimer-BODIPY 12a were studied to verify the ability of BODIPY to retain its excitation/emission properties in commonly used biological buffers. Maximal absorption and emission bands were found at 527 and 541 nm, respectively, with a quantum yield of fluorescence emission of 0.54 in no deaerated solution (Figure S3). The deactivation half-life of the fluorescent excited state was 7.85 ns (Figure S4). The dendrimer-BODIPY 12a retained its luminescent properties in PBS solution for almost a week. This analysis demonstrates that the fluorescent properties of BODIPY dye were not modified by CuAAC, by the dendrimeric moiety or by the saline-PBS environment, and could thus be employed in in vitro assays retaining all its fluorescent properties (see Table S1). We then investigated the ability of the glycodendrons to bind to the desired receptor, DC-SIGN. A titration of glycodendrons coated overnight on an ELISA plate and binding to a DCSIGN/Fc chimera was tested using a peroxidase-labeled goat antihuman Fc antibody for detection. As shown in Figure 1, both the 12a (Man) and 12b (Fuc) glycodendrons bound DCSIGN with similar efficiencies, while the binding of the 12c (Maltotriose) glycodendron was negligible. The DC-SIGN/Fc chimera is a construct carrying two units of the carbohydrate recognition domain of DC-SIGN assembled on to the Fc domain of human IgG1. However, DC-SIGN is normally found as a tetramer on the membrane of DCs.67 Therefore, we also tested binding of our glycodendrons on cells expressing DC-SIGN. Since DC-SIGN is a very efficient internalization receptor59 and we wanted to test binding at physiological conditions, we made use of a cell line transfected with a DC-SIGN mutant (DC-SIGNLL/AA)59 that is unable to mediate internalization. Cells were exposed to increasing concentrations of glycodendrons, incubated at 37 °C for 60 min, washed, and fixed. Fluorescence was then measured by flow cytometry. Results (Figure 2B) confirm a similar binding efficiency for both the 12a and 12b glycodendrons, while the binding of the maltotriose glycodendron 12c remained low. Moreover, addition of the calcium chelator EGTA, the competitive inhibitor mannan, or the blocking antibody AZN-D1 decreased binding of the 12a and 12b glycodendrons to the levels of the 12c glycodendron (data not

Scheme 3. Synthesis of Propargyl-BODIPY 11

as previously described.55 Propargyl-BODIPY dye 11 was prepared through the condensation of the benzaldehyde derivative 10 with an excess of 2,4-dimethyl-3-ethylpyrrole in the presence of a catalytic amount of trifluoroacetic acid (TFA), followed by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and treatment with BF3.·Et2O in the presence of triethylamine (TEA). After purification through chromatography on silica gel, the fluorophore was obtained in 18% yield. The fluorescent properties of propargyl-BODIPY dye 11 were similar to that previously described for this type of fluorescent chromophore. In dichloromethane, where it is soluble, it showed an absorption maximum to 526 nm and a fluorescent emission to 540 nm with fluorescence quantum yield of 0.72 (Figure S1). The decay could be well adjusted to a monoexponential profile with a deactivation half-life of 6.67 ns, which is in the order of magnitude expected for BODIPY dyes66 (Figure S2). Once, the glycodendrons and the propargyl-BODIPY derivative were prepared, the last step was the conjugation using 0.8 equiv of CuBr and TBTA at 60 °C under microwave irradiation, yielding the corresponding glycodendrons 12 with the fluorescence tag. In the case of the glycodendron with maltotriose, a first step consisting on the sugar deprotection was performed with K2CO3 in a mixture of methanol:water:THF (1:1:1). In the particular case of maltotriose nonavalent dendron 8c, the coupling with BODIPY derivative 11 was optimized using CuSO4 as a copper source, sodium ascorbate, and TBTA, at 60 °C by microwave irradiation affording the glycodendron 12c in 64% yield after purification by sephadex chromatography. The photophysical properties of the dendrimer-BODIPY 12a (assuming that the other glycodendrons should behave in a Scheme 4. Preparation of BODIPY Tagged Dendrons 12a−c

F


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early endosomes or lysosomes, suggesting that this compound was internalized by a nonspecific mechanism that results in very poor targeting to the lysosomes. It has also been shown that DC-SIGN is a signaling receptor.68,69 Moreover, signaling via DC-SIGN appears to be dependent on the glycan type.69 The signaling associated with DC-SIGN modulates the signaling of toll-like receptors. We therefore tested whether our glycodendrons were able to modulate LPS-induced maturation and cytokine responses on DCs. Surprisingly, none of the compounds was able to neither induce maturation nor modulate LPS-induced maturation (Figure 3A). Also, none of the glycodendrons induced cytokine responses and were not able to modulate the signaling induced by LPS, as shown for IL-6, IL-10, and TNFα (Figure 3B), but also for IL-12p35 and IL-12p40 by real-time PCR (data not shown).

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DISCUSSION The design of well-defined compounds as carriers to internalize peptide antigens into DCs is a topic of interest due to the potential applications of these systems as synthetic vaccines. The presence of mannosyl units on these carriers has been proved as a very powerful strategy to improve the uptake of these systems by DCs. The position of these mannosyl ligands on the carrier can influence notably the final output of these systems by a nonefficient presentation of the key peptidic epitope. For this reason, the design of systems with a full control of the structure is needed. Dendrimers are very interesting scaffolds for a multivalent presentation of carbohydrates. The stepwise synthesis of these molecules guarantees the monodispersity and a full control over the structure of these molecules. We have approached the synthesis of glycodendrons using a very efficient and straightforward convergent strategy based on the use of a CuAAC reaction. This strategy provides dendritic compounds with several copies of carbohydrates on its surface and with an adequate functionalization at the focal position for conjugation of molecules of interest such as immunogenic peptides. The synthetic strategy and the relatively small size of these glycodendritic compounds make this approach very attractive and permit the preparation of these carrier molecules in a reasonable scale for biological applications. The application of these glycodendrons 8 as carrier molecules is based on the possibility that these molecules interact efficiently with a receptor present at the surface of DCs, allowing the effective uptake of the molecules. Our previous experience70−75 with this type of glycodendrimers prompt us to consider this second-generation glycodendrons as adequate systems to interact with DC-SIGN, an important lectin present at the surface of DCs able to recognize mannosylated and fucosylated glycoconjugates. Also, this lectin is able to internalize pathogens into the DCs for degradation and activation of an immune response. With the aim to demonstrate that our glycodendrons were able to target DCs through the binding to this receptor, we designed a series of in vitro experiments based on ELISA tests. Maltotriose has been used as a negative control because is not a ligand for DC-SIGN. ELISA assays have clearly demonstrated that mannosylated (8a) and fucosylated (8b) glycodendrons interact efficiently with this lectin, but maltotriose derivative (8c) was not able to bind this receptor. These experiments were performed with an isolated lectin and also with whole cells expressing this lectin on the surface. In these later assays, a

Figure 1. 12a (Man) and 12b (Fuc) glycodendrons bind to DC-SIGN with similar affinity. A titration of glycodendrons was coated onto ELISA plates, washed, blocked with BSA, and incubated with DCSIGN/Fc for 2 h. After several washing steps, the amount of DCSIGN bound to the glycodendrons was measured with the help of a secondary antibody (peroxidase-labeled goat antihuman Fc). The binding curve for both 12a and 12b was almost identical, suggesting similar affinities for the receptor.

shown). None of the inhibitors affected the binding of the 12c glycodendron. These results indicate that the 12a and 12b glycodendrons bind specifically and very efficiently to DCSIGN, whereas the 12c glycodendron does not, and the binding observed could be attributed to nonspecific interactions. To study if our glycodendrimers mediated not only binding but also internalization, we then used the same cell line, but expressing a full-length variant of DC-SIGN. As shown in Figure 2C, similar incubation conditions resulted in a large increase in the fluorescence signal, suggesting that DC-SIGN efficiently internalized our compounds. Both cell lines K562/ DC-SIGN and K562/DC-SIGNLL/AA exhibited comparable levels of DC-SIGN expression (Figure 2A). Internalization was confirmed using imaging flow cytometry (Figure 2C). Further, we investigated the fate of the internalized glycodendrons, also by imaging flow cytometry. For these assays, we used monocyte-derived DCs, the goldstandard model for DCs in vitro. First, we investigated the binding and internalization of our compounds in these cells and were able to confirm that it occurred with similar specificity and magnitude to that observed in K562/DC-SIGN (Figure 2D,E). DCs were incubated with our compounds for a period of 30 min at 4 °C to allow the binding of the glycodendrons, and cells were washed in ice-cold PBS to remove excess glycodendron from the culture supernatant. Cells were then incubated at 37 °C for a period of 120 min, fixed, permeabilized, and stained with antibodies against the early endosome marker EEA1 and the late endosome/lysosomal marker LAMP1 (Figure 2F). The percentage of cells showing a high internalization and colocalization with these markers indicates that the 12a and 12b glycodendrons are efficiently routed toward the lysosomes (Figure 2G). Since binding could be completely abolished by calcium chelation, a competitive inhibitor or a blocking antibody, we can conclude that the 12a and 12b glycodendrimers target the lysosomes in a DC-SIGN-dependent pathway. Even though the 12c glycodendron was also internalized, there was hardly any colocalization with either G


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Figure 2. Binding of glycodendrons 12a (Man) and 12b (Fuc) leads to internalization and routing to early endosomes and lysosomes. (A) The expression level of DC-SIGN on K562/DC-SIGN and K562/DC-SIGNLL/AA was comparable. (B) Binding of dendrons was measured on K562/DCSIGNLL/AA cells. (C) Binding and uptake of dendrons was measured on K562/DC-SIGN cells. (D,E) DCs were incubated with 12a, 12b, or 12c at either 4 or 37 °C for 2 h. Cells were then washed and measured by imaging flow cytometry. The total BODIPY signal intensity (D) and the internalization score of each dendron (E) are depicted. (F) Scatter plot showing the internalization score versus the colocalization with LAMP1 of 12c. A gate was drawn to highlight a population of cells with a high colocalization and internalization score. (G) The internalization/colocalization scores for the early endosomal marker EEA1 (open bars) and the lysosomal marker LAMP1 (closed bars) calculated as shown in panel F.

introduced a fluorescence tag in our glycodendrons using as chromophore a BODIPY derivative. The conjugation was performed using again the CuAAC approach, which provided a very stable linkage between the glycodendron and the chromophore. The use of a BODIPY as a fluorescence tag was based on its interesting photophysical and photochemical properties, which were conserved on the conjugated system and under the experimental conditions. An important issue was the noninfluence on the fluorescence properties of this chromophore with the variation of pH. This fact is crucial for the analysis of the internalization process taking into account the

mutant DC-SIGN (unable to be internalized) was used to avoid the interference of internalization processes. The use of mannan (a very efficient ligand for DC-SIGN), EGTA (which removes calcium and inhibits the recognition process by this C-type lectin), and a specific antibody for the carbohydrate-recognition domain (CRD) of DC-SIGN demonstrated that the recognition process of our glycodendrons took place through DC-SIGN in a calcium-dependent manner and through the carbohydrates present in the glycodendrons. Once this interaction was confirmed, the next step was the analysis of the internalization process. For this aim we H


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Figure 3. Effects of glycodendrons on the activation and maturation of DCs. (A) DCs were incubated with compounds 12a, 12b, or 12c in the presence or absence of LPS for 18 h. DCs were then stained for the costimulatory molecule CD86. (B) DCs were incubated with either LPS, 12a, 12b, or 12c in the presence or absence of polymixin B. The bargraph shows the ratio of inhibition in CD86 upregulation. (C) DCs were incubated with compounds 12a, 12b, or 12c in the presence or absence of LPS for 18 h. Supernatants were assayed for the presence of IL-6, TNFα, and IL-10.

as ideal candidates for antigen delivery without the induction of unwanted cytokine responses.

differences on pH into cellular compartments. The pH in early endosomes is more acidic that in late endosomes. Using flow cytometry, we have demonstrated that glycodendrons 12a−b internalized efficiently into monocyte derived DCs at physiological temperature by a receptor-dependent mechanism. On the other hand, glycodendron 12c with maltotrioses was internalized poorly probably by a nonspecific endocytotic pathway. These results confirmed the potential application of these glycodendrons as carrier molecules to internalize antigens into DCs. This fact opens the door for their use to develop synthetic vaccines. For this particular application, it is important to know the intracellular routing of these molecules in DCs. This is classically done by investigating the colocalization of the targeting molecule of interest with monoclonal antibodies against specific intracellular compartments involved in antigenprocessing and presentation, and in the modulation of the immune response. The use of markers for particular expressed molecules in early endosomes (EEA1) and lysosomes (LAMP 1) showed that glycodendrons 12a and 12b were colocalized with lysosomes. Since MHC-II presentation in DCs involves the fusion of lysosomes with MHC-II-containing multivesicular bodies,76 we concluded that antigens coupled to glycodendrons could efficiently be presented on MHC-II complexes. Finally, we evaluated the capacity of glycodendrons to induce the maturation of DCs in the presence or absence of a known DC maturation stimulus such as the TLR4 ligand LPS. None of the glycodendrons tested show any activity to induce or modulate maturation or signaling. Although DC-SIGN ligation has previously been reported to modulate the signaling of TLR4,68,69 the lack of effects reported here could be related to a poor capacity of the glycodendrons to cross-link receptors at the membrane and, therefore, would render these compounds

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CONCLUSIONS

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ASSOCIATED CONTENT

In this work we have synthesized relatively simple and efficiently constructed glycodendrons by a convergent strategy based on CuAAC reactions. These glycodendrons, containing up to nine copies of carbohydrate ligands (mannoses or fucoses) interact efficiently with the DC-SIGN receptor at the surface of DCs. These glycodendrons have been tagged with a BODIPY chromophore to demonstrate the uptake process into DCs via a receptor-dependent mechanism. Glycodendrons are stored within late endosomes as probed by colocalization with LAMP1. These mannosylated or fucosylated compounds are not able to induce DCs maturation neither expression of cytokines at the concentration tested. Besides this fact, they can be considered as interesting vectors to internalize into target cells biomolecules of interest such as immunogenic peptides that can be conjugated to the focal position of these glycodendrons. We are envisaging the possibility to use these kind of molecules as synthetic vaccines.

S Supporting Information *

Synthetic details and characterization of compounds 3c, 4b−c, 5b−c, 7b−c, 8b−c, 9, 10,55 11, and 12a−c; absorbance, emission spectra, and Fluorescence lifetime of BODIPY and 12a; and NMR and MS spectra of compounds 8a−c and 11 have been supplied as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.” I


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AUTHOR INFORMATION

Corresponding Author

*Tel: + 34 954489568; FAX: +34 954460565; e-mail: javier. [email protected]. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge the financial support by the MICINN of Spain CTQ2008-01694, CTQ2010-20303 and CTQ2011-23410/BQU, the EU RTN CARMUSYS (PITNGA-2008-213592), and the European FEDER funds. J.J.G.-V. was supported by VENI NWO-ALW (Grant 863.08.020) and Astma Fonds (3.2.10.040).

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