Cellular Uptake of Gold Nanoparticles Bearing HIV gp120

Article pubs.acs.org/bc

Cellular Uptake of Gold Nanoparticles Bearing HIV gp120 Oligomannosides Blanca Arnáiz,†,‡ Olga Martínez-Á vila,† Juan M. Falcon-Perez,§,∥ and Soledad Penadés*,†,‡ †

Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE, ‡Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), P° de Miramón 182, 20009 San Sebastian, Spain § IKERBASQUE, Basque Foundation for Science; ∥Metabolomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park Bldg 801-A, Derio, 48160, Bizkaia, Spain S Supporting Information *

ABSTRACT: Dendritic cells are the most potent of the professional antigen-presenting cells which display a pivotal role in the generation and regulation of adaptive immune responses against HIV-1. The migratory nature of dendritic cells is subverted by HIV-1 to gain access to lymph nodes where viral replication occurs. Dendritic cells express several calcium-dependent C-type lectin receptors including dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), which constitute a major receptor for HIV-1. DC-SIGN recognizes N-linked high-mannose glycan clusters on HIV gp120 through multivalent and Ca2+-dependent protein−carbohydrate interactions. Therefore, mimicking the cluster presentation of oligomannosides from the virus surface is a strategic approach for carbohydrate-based microbicides. We have shown that gold nanoparticles (mannoGNPs) displaying multiple copies of structural motifs (di-, tri-, tetra-, penta-, or heptaoligomanosides) of the N-linked high-mannose glycan of viral gp120 are efficient inhibitors of DC-SIGN-mediated transinfection of human T cells. We have now prepared the corresponding fluorescent-labeled glyconanoparticles (FITCmannoGNPs) and studied their uptake by DC-SIGN expressing Burkitt lymphoma cells (Raji DC-SIGN cell line) and monocytederived immature dendritic cells (iDCs) by flow cytometry and confocal laser scanning microscopy. We demonstrate that the 1.8 nm oligomannoside coated nanoparticles are endocytosed following both DC-SIGN-dependent and -independent pathways and part of them colocalize with DC-SIGN in early endosomes. The blocking and sequestration of DC-SIGN receptors by mannoGNPs could explain their ability to inhibit HIV-1 trans-infection of human T cells in vitro.

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INTRODUCTION In the fight against the transmission of the human immunodeficiency virus (HIV), extraordinary efforts have been made to prevent (vaccines and microbicides) or eliminate infection (anti-retrovirals, therapeutic vaccines). None of these strategies have yet eradicated HIV infection or prevented transmission. A renewed effort is needed to understand the molecular basis of the complex mechanism of virus infection.1 The recent development of biofunctional nanoparticles and their applications in biomedicine opens new opportunities to address this problem. Among the early targets of HIV-1 are immature dendritic cells (iDCs) and Langerhans cells in mucosal epithelium, and CD4+CCR5+ T cells and macrophages in the subepithelium.2,3 Some studies point to iDCs as the main migratory cell populations involved in HIV-1 disseminations from intravaginal transmission.4−6 iDCs, especially those in epithelia, can recognize, bind, and ingest whole pathogens and/or their related antigens, transporting them between peripheral and lymphoid tissues. Through subversion of the migration mechanisms of DCs, HIV-1 gains access to CD4+ lymphocytes in lymphoid tissue through the so-called trans-infection mechanism and enters these cells through a series of sequential © 2012 American Chemical Society

interactions between the envelope glycoprotein (Env) and cellular receptor CD4 and coreceptors CCR5 or CXCR4. The involvement of iDCs in the transfer of HIV-1 to CD4+ lymphocytes has been described in two distinct phases. First, iDCs divert virus from the endolysosomal pathway (clathrincoated vesicles) to the DC-T cell synapse (trans-infection). This phase is temporally limited by the degradation of significant amounts of virus within 24 h postinfection. Second, residual virus replicates by “de novo production” and is transferred from iDCs to CD4+T cells (cis-infection).7 Other studies postulate that the vast majority of virions are transmitted in trans originating from the plasma membrane rather than from intracellular vesicles.8 DCs are equipped with C-type lectin receptors (CLRs) involved in HIV-1 capture such as dendritic-cell specific intercellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN; CD209) and mannose receptor (MR; CD206).9,10 CLRs are characterized by one or several carbohydrate recognition domains (CRD) that interact in a Received: December 13, 2011 Revised: March 2, 2012 Published: March 21, 2012 814

dx.doi.org/10.1021/bc200663r | Bioconjugate Chem. 2012, 23, 814−825

Bioconjugate Chemistry

Article

Figure 1. (A) High-mannose undecasaccharide of viral gp120. (B) Di-, tri-, tetra-, penta-, and hepta-oligomannoside conjugates, structural motifs of the gp120 high-mannose glycan, used for the preparation of glyconanoparticles. (C) Schematic representation of fluorescein-labeled oligomannoside gold nanoparticles (FITC-mannoGNPs). [a] The numbers indicate the percentage of mannose oligosaccharides on GNPs, with the rest being the stealthy component 5-mercaptopentyl β-D-glucopyranoside (GlcC5S) and fluorescein conjugates.

competition with the virus.31 To investigate this hypothesis, we have now prepared fluorescently labeled analogues of the mannoGNPs (FITC-mannoGNPs) bearing 50% density of di-, tri-, tetra-, penta-, or heptamannosides (Figure 1) and monitored their binding and entry into DC-SIGN expressing cells by flow cytometry (FC) and confocal laser scanning microscopy (CLSM).

calcium-dependent manner. DC-SIGN has a single CRD which specifically recognizes high-mannose clusters on gp120.11 The HIV-1 glycoprotein gp120 is highly glycosylated with highmannose type glycans.12−15 The high-mannose glycans (Figure 1) are arranged in clusters, which mediate virus attachment to DCs through multivalent carbohydrate−lectin interactions.16,17 Mimicking the cluster presentation of high-mannose glycans on the virus surface is a strategy for the generation of carbohydrate-based antiviral agents. Approaches mimicking the multivalent presentation of high-mannose oligosaccharides on proteins,18 peptides,19 dendrimers,20,21 polymers,22 liposomes,23 microspheres,24 and nanoparticles,25,26 as scaffolds, are emerging for the development of potent and selective inhibitors with potential applications as vaginal microbicides and HIV vaccines. Among these scaffolds, gold nanoparticles offer additional advantageous features, as they allow display of a large number of carbohydrates with a high density and multifunctionality by simultaneous incorporation, in a single gold cluster, of different ligands in a controlled way.27 To target the DC-SIGN receptor on DCs, we prepared a small library of glyconanoparticles bearing multiple copies of oligosaccharidic motifs of the N-linked high-mannose glycan of viral gp120 (mannoGNPs).28 These GNPs mimic the cluster presentation of the high-mannose glycans on gp120. The mannoGNPs efficiently inhibit DC-SIGN mediated transinfection of human T-cells in a cellular assay that mimics the natural route of virus transmission from DCs to Tlymphocyte.29 DC-SIGN-mediated uptake of HIV is required for trans-enhancement of T cell infection.30 We hypothesized that the ability of mannoGNPs to inhibit HIV-1 trans-infection of human T cells could be explained by binding and sequestration of DC-SIGN receptor of dendritic cells in

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EXPERIMENTAL PROCEDURES Reagents and Methods. Reagent-grade solvents and chemicals were used without further purification. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium borohydride (NaBH4), and ethylene diaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich. Chloroauric acid was purchased from Strem Chemicals. The PBS buffer was purchased in tablets and prepared following manufacturer procedures (Sigma-Aldrich) corresponding to 10 mM phosphate buffer containing 137 mM NaCl and 2.7 mM KCl at pH 7.4. UV−vis spectra were carried out with a Beckman Coulter DU 800 spectrometer. 1H NMR spectra were recorded in D2O on a Bruker AVANCE (500 MHz) spectrometer. Chemical shifts (δ) are given in ppm relative to the residual signal of D2O. For transmission electron microscopy (TEM) examinations, a single drop (10 μL) of the aqueous solution (ca. 0.1 mg/mL in Milli-Q water) of GNPs was placed onto a copper grid coated with a carbon film (Electron Microscopy Sciences). The grid was left to dry in air for several hours at room temperature. TEM analysis was carried out in a Philips JEOL JEM-2100F working at 200 kV. Milli-Q reagent-grade water was used in all experiments. Fluorescein conjugate (FITC) was prepared as reported.32 5815



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Mercaptopentyl β-D-glucopyranoside (GlcC5S) and the di- (D), tri- (T), tetra- (Te), penta- (P), and hepta- (H) mannoside conjugates described in Figure 1 were synthesized as previously reported.28 General Procedure for the Preparation of Fluorescent Gold manno-Glyconanoparticles. The fluorescent di-, tri-, tetra-, penta-, and heptamannosylated (FITC-D50, FITC-T50, FITC-Te50, FITC-P50, and FITC-H50) GNPs were synthesized following a modified protocol.28,32 A solution (0.012 M) of a 50:45:5 mixture of the oligomannoside conjugate (D, T, Te, P, or H), glucose conjugate (GlcC5S), and fluorescein conjugate (FITC) in methanol/water (3:1) was added (3 equiv) to a solution of tetrachloroauric acid (0.025 M, 1 equiv) in water. An aqueous solution of NaBH4 (1 M, 22 equiv) was then added in four portions, with rapid shaking. The black suspension formed was shaken for an additional 2 h at 25 °C, and the supernatant was then removed. The residue was dissolved in the minimum amount of Milli-Q water, and the solution was loaded into 5−10 cm segments of SnakeSkin pleated dialysis tubing (Pierce, 3500 MWCO), placed in a 3 L beaker of water, and stirred slowly, replacing with fresh water every 3−4 h over the course of 72 h. The dialyzed solution was lyophilized to afford the GNPs (see also Supporting Information). 1H NMR spectra of the glycoconjugate mixture used for the GNP synthesis and of the products recovered from the supernatant after GNP formation were recorded to confirm the ratio of the ligands attached in the GNPs. The particle size distribution of the gold nanoparticles was evaluated from several TEM micrographs by means of an automatic image analyzer. The average diameter and the number of gold atoms of the GNPs were deduced as reported.28,32 Fluorescein content on GNPs was evaluated by interpolation from a calibration curve of commercial fluorescein isothiocyanate. The fluorescence of each FITC-mannoGNP was measured in quadruplicates at λem 530 nm (λex 485 nm) with a plate reader (GENios Pro, TECAN). These values were taken as reference to normalize the fluorescence of cells after incubation with GNPs. Nanoparticle solutions were freshly prepared (Milli Q sterile water) and sterilized (Minisart filters 0.20 μm, Sartorius) before using in cell assays. For statistical calculations of the mean fluorescence values by the FACSDiva software of the Canto II flow cytometer (BD biosciences), fluorescent cells were gated for live populations, homogeneous in size (side scatter or SSC) and granularity (forward scatter or FSC). Cell Lines. The Raji line of lymphoblast-like cells, established from a Burkitt’s lymphoma, and same line stably expressing DC-SIGN were a generous gift from Dr. J. Alcami ́ (Instituto de Salud Carlos III, Madrid). This cell line was grown in RPMI-1640 medium supplemented with 10% fetal calf serum. Media also contained 2 mM L-glutamine and streptomycin/penicillin (100 U/mL penicillin and 100 μg/ mL streptomycin). Cells were subcultured following ATCC recommendations. Generation of Human Immature DCs. Immature DCs were obtained by differentiation of monocytes isolated from buffy coat packs from healthy donors (Basque Center for Transfusions and Human Tissues, San Sebastian, Spain). Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Paque Premium (GE, Heathcare, Sweden) density gradient centrifugation. Monocytes were isolated by positive selection of CD14+ cells using CD14 microbeads (Miltenyi Biotec, Germany) and cultured for 6 days in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% low

endotoxin fetal bovine serum (PAN Biotech Gmbh, Germany), 2 mM L-glutamine, 50 U/mL penicillin/streptomycin, 2 μg/mL amphotericin B, 1000 U/mL recombinant human IL-4 (Becton Dickinson), and 1000 U/mL granulocyte macrophage colony stimulating factor (GM-CSF). Medium and cytokines were replaced every 2 days. At day 6, the resulting cells displayed the characteristic phenotype;33 moderate levels of CD14, MHCII (HLA-DR), and DC-SIGN, high levels of CD11c, and negligible CD80 as determined by flow cytometry (SI Figure S11A and B). Analysis of Surface Receptor Expression. Cell surface receptor expression was analyzed in a BD Canto II flow cytometer (Becton Dickinson). Cells were washed in PBS, blocked for 30 min at 4 °C with 10% low-endotoxin heatinactivated FBS (Fetal Bovine Serum, Biochrom AG) and incubated in ice for 30 min in staining buffer (1% FBS, 0.09% sodium azide, PBS) containing saturation concentrations of the phycoerythrin(PE)-labeled anti-CD80, anti-DC-SIGN, antiCCR5, and CXCR4 mAb; APC-labeled HLA-DR and CD83 mAb; PerCP-cy5.5-labeled CD14 mAb, and PE-cy7-labeled CD11c mAb. As control, isotype-matched fluorophore-matched mouse mAb were used. Before analysis, cells were fixed in PBS containing 4% paraformaldehyde for 15 min at 4 °C, washed twice, and resuspended in staining buffer. A gate (M1) that excluded the isotypic control signal determined the percentage of positive cells. The mean fluorescence intensity was determined for those cells stained with the specific marker antibody. All the antibodies were purchased from BD biosciences. Detachment of DCs from the flask immediately prior to each experiment was committed by tapping or harsh flushing. ELISA for Secretion of IL10 and IL-12p40. To detect IL12p40 and IL-10 production by DCs, 96-well flat-bottom plates were coated for 12−15 h with 4 μg/mL antihuman IL-12p40 antibody or 2 μg/mL antihuman IL-10 antibody, respectively. The plates were blocked with PBS containing bovine serum albumin (BSA, 1%), sucrose (5%), and sodium azide (0.05%) for 2 h and incubated for 12−15 h with 100 μL of human recombinant IL-12p40 (hrIL-12p40), IL-10 (hrIL10) standards, or supernatants of iDCs (104) incubated with LPS (1 μg/mL), gp120 (35 ng/mL), Te50GNPs (3500 ng/mL, 350 ng/mL, and 35 ng/mL) or media alone for 24 h at 37 °C. Following the incubation, biotinylated anti-IL12p40 (175 ng/mL) or biotinylated anti-IL-10 (300 ng/mL) antibody was added, respectively, and the incubation continued for 2 h. Levels of IL12p40 and IL-10 were detected using streptavidin−horseradish peroxidase (SA-HRP, 1:2500) in PBS and its substrate pnitrophenyl phosphate according to manufacturer’s instructions. The reaction was stopped with 50 μL of H2SO4. Absorbance was measured at 492 nm. All the incubations and reactions were performed at room temperature. After each incubation, all the wells were washed three times with distilled water containing NaCl (150 mM) and Tween-20 (0.05%).The antibodies and standards (DuoSet human IL-12p40) used were purchased from R&D Systems. Cellular Uptake of FITC-mannoGNPs. For internalization assays, 105 cells (Raji, Raji DC-SIGN, and iDCs) were incubated in 300 μL of supplemented medium (RPMI-1640) containing 25 μg/mL of FITC-mannoGNPs (FITC-D50, FITC-T50, FITC-Te50, FITC-P50, and FITC-H50) or media alone (untreated) for 3 h at 37 °C, 5% CO2. Unbound mannoGNPs were removed by two washes with cold PBS before FC analysis. Raji DC-SIGN cells incubated with FITC816



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addition of 3.8 mL thiourea (200 μg/mL) in HCl 1% for analysis in an Agilent 7500 CE. Localization of Intracellular FITC-Te50GNP. Confocal microscopy was performed on a Zeiss LSM 510 META microscope equipped with 63× magnification oil lens. iDCs were incubated with FITC-Te50GNPs (25 μg/mL) in media. Following 3 h incubation, 3 × 10 5 cells were deposited on polylysine-coated coverslips and left for 1 h at 37 °C to allow adherence. Cells were washed three times with PBS at 37 °C, fixed with 4% paraformaldehyde PBS (37 °C) for 15 min and washed again three times with PBS. Cells were permeabilized with PBS containing 0.05% saponin and 10% BSA for 10 min at room temperature and then blocked with 3% BSA in PBS at 4 °C overnight. For double immunostaining, permeabilized iDCs were incubated at 37 °C for 2 h with both rabbit antihuman DC-SIGN mAb (Abcam, 10 μg/mL) and mouse antihuman mAb against LAMP-1 (BD Pharmigen, 0.5 μg/mL), EEA-1 (BD Pharmigen, 5 μg/mL), HLA-DP, DQ&DR (Ancell, 50 μg/ mL), Cav-1 (BD Pharmigen, 5 μg/mL), or Clathrin (BD Pharmigen, 5 μg/mL) in 1% BSA PBS buffer. After extensive washes with 0.5% Tween-20 in PBS, cells were first incubated with 1% BSA in PBS containing secondary goat antirabbit antibody−Alexa Fluor 405 (Invitrogen, 5 μg/mL) at 37 °C for 1 h, washed three times with 0.5% Tween-20 PBS and then incubated 1 h with rabbit antimouse antibody−Alexa Fluor 594 (Invitrogen, 5 μg/mL) and washed again. In an attempt to palliate the low pH of some organelles that leads to FITC fluorescence quenching, labeled iDCs were mounted in FluorSave oil (Merck, pH 10.5−10.7) and examined under the confocal microscope with a 63× objective at excitation wavelengths of 488, 405, and 561 nm for fluorescein, Alexa Fluor 405, and Alexa Fluor 594, respectively. Images of cells photographed with CLSM were analyzed with Zeiss LSM Image Browser or ImageJ software. Quantification Analysis of Colocalization. For intensity quantification of each color along a defined line, two-color overlays were analyzed with the Zen 2007 software and generated fluorescence intensity histograms. Colocalization coefficients displayed in Table S1 were obtained by ImageJ software analysis of pairs of 8-bit images (488/561 nm or 405/ 488 nm) using automatic threshold and background correction.35 Quantification of the degree of colocalization of the different vesicular markers was carried out as previously reported using imaging software.36,37 A total of 1413 fluorescein-positive objects with a mean area of 0.62 μm2, each of them corresponding to independent vesicles, were detected in the 90 iDCs (mean = 15.7 and SD = 8.9) and processed to evaluate the colocalization degree with DC-SIGN and EEA1 positive compartments by using the imaging software Volocity v 5.5. (Perkin-Elmer).

Te50GNPs were stained with DAPI (100 ng/mL) for 20 min, washed with PBS, and deposited on a coverslip for confocal imaging. For inhibition of energy-dependent uptake, cells were incubated for 3 h at 4 °C, 5% CO2. For receptor digestion, cells were incubated with 1 mg/mL trypsin/EDTA PBS (SigmaAldrich) at 37 °C for 15 min previous to FC analysis. For inhibition assays, cells were preincubated with the corresponding inhibitors (EDTA or anti-DC-SIGN IgG1 mAb) in supplemented RPMI-1640 medium for 30 min at 37 °C, 5% CO2, before addition of FITC-mannoGNPs. The final concentrations of the inhibitors used were 50 mM of EDTA34 (Sigma), 10 μg/mL of anti-DC-SIGN IgG1 mAb (clone AZND1, Beckman Coulter, France), and 10 μg/mL of anti-immunoglobulin IgG1 isotype mAb as a control (BD biosciences). GNP uptake is expressed as a ratio between the mean fluorescence intensity of cells treated with GNPs and untreated samples (fluorescence background) and normalized to the fluorescence content of GNPs (relative fluorescence intensity). GNP uptake inhibition is presented as the percentage of the mean cell fluorescence signal in the presence of EDTA, AZND1, or isotype control to the signal in its absence. For each acquisition, the autofluorescence was eliminated from the fluorescent signal up to a residual 10%. Cells incubated in the absence of FITC-mannoGNPs were set in the cytometer to give the lowest signal in the histogram (first decade, around 50 U.A). Then, the cells incubated in the presence of FITC-mannoGNPs were acquired under the same voltage conditions. Kinetics of FITC-Te50GNPs Uptake. For uptake time course determination, cells were incubated with FITCTe50GNP (25 μg/mL) for up to 11 h. Cell aliquots (105) were washed with cold PBS at different incubation times (2.5, 5, 10, 30 min; and 1, 1.5, 2, 2.5, 3, 4, 4.5, 5, 5.5, 6, 6.5, 7.5, 9, 10, and 11 h) and the internalized FITC-Te50GNP measured by FC. For FITC detection in the FC, all the samples were excited with a 488 nm laser, and both a 502 nm long-pass and a 530/30 nm band-pass filter were used. At 2 h incubation time, the cell sample was split, and one part was washed with PBS and incubated in fresh media for up to 9 additional hours. At times 2.5, 3, 4, 4.5, 5, 5.5, 6, 6.5, 7.5, 9, 10, and 11 h, a cell aliquot was washed and resuspended in cold PBS and fluorescence decrease measured by FC. The fluorescence time course was plotted as time versus relative fluorescence intensity obtained by dividing the mean fluorescence intensities of cells treated with FITC-Te50GNP by the untreated samples and normalized with the fluorescence content of FITC-Te50GNPs. The fluorescence time course of iDCs was also followed by CLSM. Cells were incubated with FITC-Te50GNPs (25 μg/ mL) and imaged at 5 min, 2.5 h, and 5 h. At 2 h incubation time, the cell sample was split, and one part was washed with PBS and incubated in fresh media for 3 h and imaged. For imaging, 3 × 105 cells were deposited at 37 °C and 5% CO2 on polylysine-coated coverslips to allow adherence. Cells were washed twice in PBS, fixed with 4% paraformaldehyde PBS for 30 min at 37 °C, and washed twice with PBS and mounted with DAPI-containing oil (Vectashield, Vector Laboratories). Gold content of cells incubated with FITC-Te50GNP for 2.5 h was determined by inductive couple plasma−mass spectrometry (ICP-MS). Cells (105) were washed with media and the cell pellet lyophilized and digested by addition of 180 μL of HNO3 and 20 μL of HCl, heated at 60 °C overnight before the

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RESULTS AND DISCUSSION Synthesis of FITC-mannoGNPs. Fluorescein-labeled oligomannoside gold nanoparticles (FITC-mannoGNPs) coated with different structural motifs of HIV-1 gp120 highmannose glycan were prepared to study their binding/ internalization into DC-SIGN expressing cells. FITC-mannoGNPs having an average density of 50% linear di-(D), tri(T), or tetra-(Te), or branched penta-(P) and hepta-(H) mannoside conjugates, 45% of 5-mercaptopentyl β-D-glucopyranoside (GlcC5S) and 5% of fluorescein conjugate (FITC) (Figure 1) were prepared and characterized following a modification of previously described methodologies.28,32 The 817



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Figure 2. Characterization of FITC-Te50GNP. (A) TEM micrograph in H2O. (B) Size distribution histogram. (C) 1H NMR spectrum in D2O. (D) UV−visible spectrum. (E) Fluorescence spectrum.

5% of the total number of glycoconjugates as determined by fluorescence (Figure 2 and SI Figures S1−S5). Binding and Uptake of FITC-mannoGNPs by Raji DCSIGN and iDCs. Application of the FITC-mannoGNPs to cells was preceded by viability assays. Incubation of monocyte derived iDCs, Raji, or Raji DC-SIGN cell lines with 100 μg/mL FITC-mannoGNPs for 24 h indicates the absence of cytotoxic effects on Raji and Raji DC-SIGN cells and moderate effects on the viability of monocyte-derived iDCs (SI Figure S6). The incorporation of fluorescein in the mannoGNPs did not induce apparent cytotoxicity when compared with the analogous nonfluorescent mannoGNPs tested at the same concentration in the Raji DC-SIGN cell line.29 Subsequently, FITCmannoGNPs at 25 μg/mL were employed safely for all the experiments described in this work. The binding of FITC-mannoGNPs by monocyte-derived iDCs or Raji and DC-SIGN stably transfected Raji cells was explored under different experimental conditions by FC. DCSIGN expression was confirmed in both Raji DC-SIGN cell line and iDCs by immunolabeling with PE-conjugated antihuman DC-SIGN antibody (SI Figure S7). FITC-mannoGNP binding and uptake was first assessed by incubation with Raji or Raji DC-SIGN cells for 3 h at 37 or 4 °C, and cellular fluorescence

average number of ligands on the gold surface is between 100 and 120. Mannoside conjugates 1−5 were synthesized by thiourea coupling of the corresponding 2-aminoethyl αoligomannosides and the isothiocyanate linker 6 followed by subsequent removal of the acetyl group, affording compounds 1−5 as disulfide derivatives.28 The fluorescein conjugate was synthesized as previously reported.32 The length and flexibility of the linker of the fluorescein conjugate were chosen in order to avoid fluorescence quenching. To control the density of oligomannosides and fluorescein on the GNPs, the GlcC5S conjugate was selected as the stealthy component because its insignificant contribution to DC-SIGN mediated trans-infection had been proven.29 An aqueous solution of tetrachloroauric acid was added to a methanol−water mixture containing the three different conjugates in the desired ratio. The resulting mixture was reduced with an excess of NaBH4, and the suspension was vigorously shaken for 2 h at 25 °C. The supernatant was removed and the residue was dissolved in Milli-Q water and purified by dialysis. The resulting FITCmannoGNPs were characterized by transmission electron microscopy (TEM), 1H NMR, UV−vis, and fluorescence spectroscopy. The average diameter of the gold core was 1.2− 2.0 nm, and the fluorescein molecules per cluster are around 818



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Figure 3. DC-SIGN receptor-mediated internalization of FITC-mannoGNPs. (A) GNPs or media alone were incubated with Raji or Raji DC-SIGN cells for 3 h at 37 or 4 °C. Raji DC-SIGN cells were treated with trypsin to distinguish internalized from cell-surface-bound GNPs. Cell fluorescence intensity was measured by flow cytometry (FC) and expressed as a ratio between the mean fluorescence intensity of cells treated with GNPs and untreated samples, and normalized to the fluorescence content of GNP (relative fluorescence intensity). (B) Confocal image of Raji DC-SIGN cells incubated with FITC-Te50GNPs (green) for 3 h at 37 °C. Nuclei were stained with DAPI (blue). Transmitted light overlay shows the delimited plasma membrane. Data are from a representative experiment carried out twice with similar results.

Figure 4. Flow cytometry of the specific FITC-mannoGNPs uptake by C-type lectins (CLRs) and DC-SIGN receptors. Uptake of GNPs (25 μg/ mL) after 3 h by Raji DC-SIGN cells (A) or iDCs (B) in the absence or presence of EDTA (50 mM). (C) Comparison of FITC-Te50GNP and FITC-T50GNP uptake in the presence of EDTA or anti-DC-SIGN IgG1 mAb (AZN-D1, 10 μg/mL), specific for DC-SIGN carbohydrate recognition domain. A control sample with an anti-IgG1 isotype mAb (10 μg/mL) is included. FITC-mannoGNPs uptake is presented as the percentage of the mean cell fluorescence signal in the presence of EDTA, AZND1, or isotype control to the signal in its absence. Values were normalized to the FITC content per mannoGNP. (D) Dot plots of the effect of 50 mM EDTA on the size (side scatter, SSC) and granularity (forward scatter, FSC) of Raji DC-SIGN cells and iDCs after a 3 h incubation. A representative experiment out of three with similar results is shown.

times above the cell autofluorescence levels (Figure 3A, Raji DC-SIGN/37 °C). FITC-Te50GNP showed a preferential uptake in comparison with the rest of the GNPs. In the absence

was detected by FC (Figure 3). In the presence of DC-SIGNexpressing cells, all FITC-mannoGNPs were efficiently internalized at 37 °C reaching fluorescence levels at least 3 819



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Figure 5. Time course of the FITC-Te50GNP uptake at 37 °C. FC analysis of the uptake of GNPs (25 μg/mL) by Raji DC-SIGN (A, filled squares) and Raji cells (A, filled triangle), and iDCs (B, filled squares). After 2 h of FITC-Te50GNP uptake, medium containing GNPs was replaced with medium alone and cultured again (empty symbols). Data are expressed as a ratio of the mean fluorescence intensity of Te50-FITC-treated cells to untreated cells. (C) CLSM analysis of iDCs after 5 min, 2.5 h, and 5 h of FITC-Te50GNP incubation and (D) 3 h after GNP removal from the media (arrows). The images are overlays of fluorescein (green) and DAPI (blue) of a single section of the Z-stacks acquired at each time point.

of DC-SIGN (Raji cells), FITC-mannoGNPs were barely internalized (Figure 3A, Raji/37 °C). The entry in Raji DCSIGN was mostly inhibited by incubation at 4 °C (Figure 3A, Raji DC-SIGN/4 °C). The incubation of cells at 4 °C has been extensively described as a method to inhibit energy-dependent internalization. Confocal imaging of Raji DC-SIGN after incubation with FITC-Te50GNPs corroborates that internalization occurs (Figure 3B). Together, these results show that the entry of fluorescently labeled oligomannoside-coated GNPs in Raji DC-SIGN is mediated by active mechanisms and, to a much less extent, by passive mechanisms. Trypsination before FC measurements (Figure 3A) showed a small reduction of fluorescence. This result indicates that a high fraction of FITCmannoGNPs is internalized via receptor-mediation, and only a small fraction remains bound to plasma membrane. In order to prove the involvement of the Ca2+-dependent CLRs in the active uptake of mannoGNPs, Raji DC-SIGN and iDC were incubated with the nanoparticles in the presence of ethylenediaminetetraacetic acid (EDTA) (Figure 4). EDTA inhibits the binding of oligosaccharides to CLRs by chelating calcium cations. Dot plots in Figure 4D show the lack of drastic effects of 50 mM EDTA on the size and granularity homogeneity of Raji DC-SIGN cells and iDCs after a 3 h incubation. The uptake of mannoGNPs by Raji DC-SIGN cells or iDCs for 3 h at 37 °C was partially inhibited by 50 mM EDTA (Figure 4A,B). The rest of the total uptake observed (∼50%) could be attributed to CLR-independent pathways. As expected, EDTA treatment did not decrease the uptake of FITC-mannoGNP by Raji cells (data not shown) indicating that CLRs are not involved. In order to investigate the role of DC-SIGN in the cellular uptake of mannoGNPs, we selected the anti-DC-SIGN mAb

AZN-D1 to block specifically the carbohydrate recognition domain (CRD) of DC-SIGN. This antibody has been shown to inhibit HIV-1 gp120/DC-SIGN interaction38 and HIV transmission to CD4+T cells.39 The fluorescently labeled FITCT50GNP and FITC-Te50GNP, whose analogues (T50GNP and Te50GNP) showed the highest inhibitory activity in the DC-SIGN mediated HIV-1 trans-infection of T-cells,29 were used as probes to test whether the uptake in Raji DC-SIGN and iDCs is dependent on the mannoside/DC-SIGN interaction. We found that the GNP entry in monocyte-derived iDCs and Raji DC-SIGN was specifically diminished by 10 μg/mL of antibody (Figure 4C). Higher concentrations of mAb AZN-D1 (15 μg/mL and 20 μg/mL) improved the inhibition levels (SI Figure S8). As control, we used an antibody of the same isotype as AZN-D1 (IgG 1), which did not inhibit the uptake of FITCT50GNP and FITC-Te50GNP (Figure 4C). The entry inhibition of the FITC-T50GNP and FITC-Te50GNP by mAb AZN-D1 in Raji DC-SIGN and iDCs and the transinfection inhibitory activity observed for both GNPs suggest that the multivalent display of the mannosides on the GNPs competes with HIV-1 for the carbohydrate recognition domain of the DC-SIGN receptor. Kinetics of the Uptake Process of FITC-Te50GNPs by iDCs, Raji, or Raji DC-SIGN cells. Cell uptake of FITCTe50GNP was monitored by FC and CLSM at different incubation times (Figure 5). iDCs, Raji, or Raji DC-SIGN cells were incubated with FITC-Te50GNP (25 μg/mL) for up to 11 h at 37 °C allowing uptake (Figure 5A,B, filled symbols). As expected, FITC-Te50GNP was efficiently internalized in Raji DC-SIGN cells and iDC. The uptake reaches a plateau (equilibrium state) at 2−2.5 h in Raji DC-SIGN cells and iDCs. This uptake was 4 times higher than in Raji cells. The 820



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Figure 6. (A) Schematic representation of FITC-Te50GNP. (B) Orthogonal view of a Z-stack. (C) Gallery view of single plane series of a Z-stack. iDCs were incubated with FITC-Te50GNP (green) for 3 h at 37 °C, plated on glass coverslips, and fixed before mounting. Nuclei were counterstained with DAPI (blue). Confocal optical 2 μm sections in 730 nm intervals taken through the whole cells show intracellular localization of FITC-Te50GNP (scale bars = 20 μm).

small uptake observed in Raji cells remains constant (Figure 5A). After 2 h incubation half of the cell sample was washed with PBS and centrifuged to remove the free FITC-Te50 nanoparticles and the cells were incubated in fresh medium for additional 9 h. Maximum fluorescence decrease is reached after 1.5 h for Raji DC-SIGN (Figure 5A, empty squares) and 5−6 h for iDCs (Figure 5B, empty squares). The decrease in iDC was 4-fold higher than the decrease in Raji DC-SIGN. The curves indicate that 80−90% of fluorescence remains in Raji DC-SIGN and 40−60% in iDCs. Confocal imaging confirmed the cellular internalization of FITC-Te50GNPs in iDCs (Figure 5C and SI Figure S9 upper panel). After removing the free GNPs, a fluorescence decrease was observed (Figure 5D). Inductively coupled plasma mass spectrometry (ICP-MS) analysis after 2.5 h uptake of FITC-Te50GNPs showed ∼90% and ∼95% of gold content inside Raji DC-SIGN and iDCs, respectively. The discrepancy between the fluorescence values and the gold content in iDCs deserves further investigation. It has been reported that the quantum yield of fluorescein is pHdependent.40 Accumulation of FITC-Te50GNPs in subcellular acidic compartments such as late endosomes could explain the loss of fluorescence signal in iDCs.41 Analysis of Intracellular Localization of FITCTe50GNP. Additional evidence of intracellular distribution of FITC-Te50GNP was supplied by confocal fluorescent optical z sections (Figure 6). As shown in Figure 6, FITC-Te50GNP shows a punctuated pattern distribution after 3 h uptake of nanoparticles. To study the intracellular compartment distribution and routing of FITC-Te50GNP in iDCs, mAbs specific for DC-SIGN, caveolin (Cav-1), clathrin, early endosome antigen 1 (EEA1), lysosomal-associated membrane protein 1 (LAMP-1), and major histocompatibility complex class II (HLA-DP, -DQ, and -DR) were used to specifically label intracellular organelles in iDCs. Some of these organelles

are thought to be potentially involved in the endocytic pathway of DC-SIGN and gp120/HIV-1.42−45 Qualitative analysis of the images using fluorescence intensity histograms did not allowed us to obtain conclusive colocalization results (SI Figure S9). As an attempt to overcome this problem, we followed a quantitative protocol based on coefficient calculations.35 Overlap coefficient according to Manders indicated that both GNPs and DC-SIGN colocalize between 60% and 70% with Cav-1, clathrin, EEA1, LAMP-1, and MHCII positive compartments. However, Pearson’s correlation coefficients (0−0.3) indicated absence of colocalization (SI Table S1). To clarify this contradiction, a comprehensive quantitative analysis using the imaging software Volocity v 5.5 (Perkin-Elmer) was carried out on iDCs loaded with FITC-Te50GNPs (Figure 7). In the 90 iDCs that were processed, a total of 1413 FITC-positive objects (GNPs) with a mean area of 0.62 μm2, each of them corresponding to independent organelles, were detected. 62% of the FITCTe50GNPs-positive objects were positive for DC-SIGN, 43% were positive for the early endosome marker, and only 36% were positive for the both markers. Apart from early endosomes, FITC-Te50GNPs were also observed in organelles labeled with the late endocytic marker, LAMP1 (SI Figure S9). Together, these results confirm that at least half of the Te50GNP-containing organelles also contain DC-SIGN receptor, and indicate that GNPs can enter the cells by binding to DC-SIGN and routing through the early endosomes similarly to gp120.7,44 Remarkably, 38% of the FITCTe50GNPs do not colocalize with the DC-SIGN receptor, which can be interpreted as DC-SIGN-independent modes of entry for GNPs could exist or that, after DC-SIGN-dependent entry occurs, GNPs and DC-SIGN follow different intracellular fates. Further investigations to differentiate between these alternatives are certainly required. 821



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Figure 7. Representative images of quantitative colocalization analysis. (A) Bright-field image of a selected representative iDC. (B). FITCTe50GNP+ objects selected by Volocity software in a single cell image with an intensity threshold established from the fluorescence background observed outside the cell. (C,D,E) Single image of FITC-Te50GNP (green), early endosomes (red), and DC-SIGN (blue). Overlay images of (F) FITC-Te50GNP and DCSIGN; (G) FITC-Te50GNP and early endosomes; (H) FITC-Te50GNP, DC-SIGN, and early endosomes; and (I) colocalization degree of FITC-Te50GNPs, with DC-SIGN receptor and the early endosomal marker EEA-1. Values represent percentages of the structures conforming to the definitions indicated in the table out of the total number of structures scored positive for FITC-Te50GNPs.

mannose glycan clusters with DC-SIGN. Thus, the most plausible explanation for the inhibitory effect of mannoGNPs in HIV-1 trans-infection29 may be their competition with the virus for binding to DC-SIGN. In an in vivo scenario, DCs are vulnerable to productive HIV-1 infection.3 Therefore, in addition to inhibiting trans-infection by binding DC-SIGN, mannoGNPs could also down-regulate other receptors (CCR5 and CXCR4) involved in the HIV infection of DCs. In order to

The multivalent oligomannoside-coated GNPs are able to enter Raji DC-SIGN and iDCs by CRL-dependent and -independent ways. We have demonstrated that GNPs incorporating D1 branch tri- and tetramannosides of the gp120 in 50% density enter partially Raji DC-SIGN and iDCs by binding specifically to DC-SIGN lectin. This indicates that multimerization of the D1 branch oligomannosides on gold nanoparticles mimics the interaction of HIV/gp120 high822



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test this possible effect, the level of DC-SIGN, CCR5, and CXCR4 on iDC plasma membrane upon 3 h treatment with FITC-Te50GNP was determined by FC. The expression of the receptors in GNP-treated cells was the same as in the untreated cells (SI Figure S10). Phenotypic characteristics and/or cytokine secretion of DC after longer incubation periods was also evaluated. iDCs incubated for 24 h with Te50GNPs (350 ng/mL and 3500 ng/mL) showed the opposite maturing effect on DC-SIGN and CD83 expression than lipopolysaccharide (LPS, 1 μg/mL), while no effect was observed on CD80 expression, or IL12p40 or IL10 secretion (SI Figure S11 B&C). Te50GNPs down-regulate the expression of CD83 in iDCs in a dose-dependent manner (SI Figure S11B) and up-regulate the expression of DC-SIGN. Te50GNPs showed a similar but more pronounced effect on CD83 and DC-SIGN expression than gp120. The results suggest a lack of maturing effect by Te50GNPs at the concentrations tested and a certain opposite effect on the iDC maturation phenotype.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Spanish Ministry of Science and Innovation (grant CTQ2011-27268), the EU (grant CHAARM), and the Department of Industry and Innovation of the Basque Country Government (grant ETORTEK). The reagent ARP971 GM-CSF Recombinant and gp120 from the HIV-1 CN54 clone (repository reference ARP683) were obtained from the Programme EVA Centre for AIDS Reagents, NIBSC, UK, supported by the EC FP6/7 Europrise Network of Excellence, AVIP and NGIN consortia, and the Bill and Melinda Gates GhRC-CAVD Project and was donated by Invitrogen. We thank Dr. J. M. Cárdenas, director of the Basque Centre for Transfusions and Human Tissue for the access to blood samples from healthy donors.

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CONCLUSIONS Gold glyconanoparticles bearing multiple copies of structural motifs of the high-mannose glycan clusters of gp120 (mannoGNPs) are internalized by C-type lectin-expressing cells. We demonstrate that entry of the 1.5−1.8-nm-sized mannoGNPs is mediated by both CLR-dependent and CLRindependent mechanisms. The internalization mediated by CLRs accounts for about 50% of the total entry in iDCs. The rest of mannoGNP seems to enter by other active mechanisms (i.e., constitutive macropinocytosis). The tetramannoside coated nanoparticle (FITC-Te50GNP) mimics the behavior of gp120 in their ability to bind to DC-SIGN and internalize into the early endosomes in iDCs. These results support our idea that the multivalent presentation of oligomannosides onto the GNP surface provides a tool for the development of HIV microbicides and vaccines. The incorporation into the GNPs of T cell peptide epitopes and/or immunostimulant adjuvants could be a strategy to break the immunotolerance promoted by high-mannose-type glycans.33 Understanding the interaction of engineered biofunctional nanoparticles with cells has implications in their design and improvement and in clarifying the impact of these nanomaterials on human and environmental health.46 Previous studies have shown that size, shape,47,48 surface modifications,49 and even sedimentation and diffusion50 play a role in cellular uptake, trafficking, and removal of nanoparticles. The great diversity in the size, shape, and surface of nanoparticles as well as the multiple portals51 that regulate the uptake by cells makes it difficult to establish a common mechanism of internalization for nanomaterials. Furthermore, for in vivo applications, the pitfalls in performing studies of endocytosis and intracellular transport of nanoparticles must be taken into account.52

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ASSOCIATED CONTENT

S Supporting Information *

Characterization data of FITC-mannoGNPs and results on cell viability. DC-SIGN expression in Raji DC-SIGN and iDC. FITC-Te50GNP intracellular distribution and quantitation, and their effect on DC-SIGN, CCR5, and CXR4 expression on iDC. Phenotype and cytokine secretion of DC before and after 24 h incubation with Te50GNPs. This material is available free of charge via the Internet at http://pubs.acs.org. 823



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Cellular Uptake of Gold Nanoparticles Bearing HIV gp120

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