Carbohydrate-Coated Fluorescent Silica Nanoparticles as Probes for

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Carbohydrate-Coated Fluorescent Silica Nanoparticles as Probes for the Galactose/3-Sulfogalactose Carbohydrate−Carbohydrate Interaction Using Model Systems and Cellular Binding Studies Jingsha Zhao,† Yuanfang Liu,‡ Hyun-Joo Park,‡ Joan M. Boggs,‡,§ and Amit Basu*,† †

Department of Chemistry, Brown University, Providence, Rhode Island 02912, United States Molecular Structure and Function Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 § Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L5 ‡

S Supporting Information *

ABSTRACT: The carbohydrates galactose and 3-sulfogalactose, found on sphingolipids in myelin, interact with each other via a carbohydrate−carbohydrate interaction (CCI). In oligodendrocytes, this interaction triggers a signaling cascade resulting in cytoskeletal rearrangements and reorganization of glycolipids and proteins at the cellular surface. These rearrangements can also be triggered by synthetic multivalent glycoconjugates. In this report, we describe the synthesis of glycan-coated silica nanoparticles and their subsequent binding to cultured oligodendrocytes and purified myelin. Fluorescent silica nanoparticles with an azidosiloxane-derived outer shell were functionalized with carbohydrates using the copper-promoted azide−alkyne cycloaddition reaction. The carbohydrate−carbohydrate interaction between galactose and 3-sulfogalactose was examined by measuring the binding of 3-sulfogalactose-containing nanoparticles to galactolipids that had been immobilized in a multiwell plate. Particle aggregation mediated by CCI was observed by TEM. The interaction of the particles with oligodendrocytes and purified myelin was examined using fluorescence microscopy, providing direct evidence for binding of galactose and 3-sulfogalactose-coated nanoparticles to oligodendrocytes and myelin fragments.

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INTRODUCTION Carbohydrate−carbohydrate interactions (CCIs) are molecular recognition events that involve the binding or association of one glycan with another.1,2 CCIs between polysaccharides mediate processes such as cellulose fibril formation and their subsequent cross-linking by xyloglucan,3 as well as proteoglycan-mediated self-association of sponge cells.4,5 Other CCIs engage cell surface glycolipids for the proper compaction of developing embryos and mediate melanoma cell adhesion to endothelia.6,7 As with much of carbohydrate recognition, CCIs are weak interactions and involve multivalent presentations of glycans. Studies of CCI in their native context are complicated by the heterogeneous environment of the cell surface, and the use of multivalent glycoconjugates as model systems have been invaluable in establishing the occurrence of several CCIs. We and others have examined the interaction between Langmuir monolayers of glycolipids and glycoconjugates such as micelles,8 glycodendrimers,9 and glycopolymers.10 Quantitative measurements of CCI-mediated binding and adhesion have been provided by surface plasmon resonance (SPR), quartz crystal microbalance, and giant vesicle micropipet experiments.11−13 Carbohydrate-coated gold nanoparticles are a versatile platform for studies of CCI, and have been used to investigate the interaction using TEM, SPR, and calorimetry.14−17 © XXXX American Chemical Society

Fluorescent nanoparticles are an attractive platform for presenting carbohydrates, as they can be used for diagnostics, sensors, and cellular imaging applications.18−23 In particular, silica nanoparticles with encapsulated fluorescent dyes offer several attractive features(i) they are readily and cheaply synthesized; (ii) silica is biocompatible; and (iii) encapsulation of fluorescent dyes by the silica matrix enhances photostability of the chromophore.24 A fourth benefit is the ability to readily modify the nanoparticle surface with ligands for subsequent studies. A variety of different methods for the functionalization of silica nanoparticles have been reported. A large number of these methods involve coating nanoparticles with an aminoterminated silane, followed by subsequent coupling to the amino group.25−27 The thiol exchange reaction of a disulfide has been used to attach oligonucleotides onto silica nanoparticles coated with a mercaptosilane.28 Pentafluorophenylazide coated silica nanoparticles can be covalently modified via photochemical generation of a nitrene.29,30 Surface chlorination with thionyl chloride has also been reported.31 Nickel nitrilotriacetic acid modified particles have been used to capture His6-tagged proteins.32 Received: November 15, 2011 Revised: April 24, 2012

A

dx.doi.org/10.1021/bc2006169 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry

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esis that the liposomal GalCer and SGalCer bind to GalCer and SGalCer in the cell membrane, Fab fragments of anti-GalCer antibodies and fumonisin B1, which inhibits glycosphingolipid synthesis, prevented liposome-induced glycolipid redistribution on the cell surface.53 Subsequent studies using galactose-coated bovine serum albumin (Gal-BSA), a commercially available glycoconjugate, as well as glycosylated nanoparticles, yielded results similar to those observed with the liposomes.54,55 It is believed that the “receptor” for SGal and Gal on the oligodendrocyte are GalCer and SGalCer, respectively, and that the glycan-presenting liposomes and BSA engage in a CCI on the cell surface that triggers transmembrane signaling and a cytoskeletal rearrangement. We envisioned that glycosylated fluorescent silica nanoparticles would provide a powerful tool for studying the CCI between Gal and SGal, since they can both be used to examine CCI with a well-defined model system, and can also be used to examine the CCI in a cellular context by directly visualizing the binding of glycoconjugates on the oligodendrocyte surface.

The copper(I) promoted azide−alkyne cycloaddition reaction33,34 is a “click” reaction35 which provides a facile and mild method for nanoparticle functionalization.36−41 The reaction is regioselective, high-yielding, compatible with a large number of functional groups, and has recently been used to attach polymers, peptides, and fluorophores to silica nanoparticles.42−46 These features make this a useful reaction for attaching carbohydrates to fluorescent silica nanoparticles. In this paper, we report the synthesis of fluorescent carbohydratecoated nanoparticles and their use in probing a CCI found in myelin. The myelin sheath is a multilayered membrane in the nervous system which encircles axons and facilitates the propagation of action potentials down the axon. Myelin membranes are formed by oligodendrocytes in the central nervous system and by Schwann cells in the peripheral nervous system. The lipid bilayer of myelin is highly enriched in glycolipidsit has been estimated that up to 40% of the outer leaflet is composed of two glycolipids, galactosylceramide (GalCer), and its 3-sulfo derivative, sulfatide (SGalCer).47 These lipids are important for proper myelination, and knockout mice that lack one or both of these exhibit abnormal myelination and various other molecular and physiological defects.48

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EXPERIMENTAL METHODS General experimental procedures are provided in the Supporting Information. Synthesis of Azide Linker-Coated Fluorescent Silica Nanoparticles (FITC-doped Si NPs, 4). 3-Aminopropyltriethoxysilane (APTS) (8 mg, 0.039 mmol) and fluorescein isothiocyanate (FITC) (15 mg, 0.039 mmol) were combined together in anhydrous DMF (1 mL) under a dry nitrogen atmosphere and stirred for 12 h. The solution was wrapped with aluminum foil during the reaction. The solution of the FITC-APTS conjugate 3 was used directly in the next step. Igepal CO-520 (240 μL) was dissolved in cyclohexane (4.5 mL) in a 20 mL vial. After stirring the solution for 10 min, NH4OH solution (120 μL) was added, and the mixture was stirred for 10 min to provide a homogeneous suspension. FITC-APTS conjugate (20 μL) was added, followed by TEOS (60 μL, 0.27 mmol) another 5 min later. In order to get a pure silica surface, TEOS (20 μL) was added after 24 h, and stirring was continued for another 24 h. To this solution was added siloxane 2 (4 mg, 0.015 mmol) and stirring was continued for another 24 h. Acetone (10 mL) was added. The mixture containing silica nanoparticles was distributed in a 20 mL centrifuge tube, and the suspension was centrifuged for 15 min. The silica nanoparticles were precipitated on the bottom, and the supernatant was discarded. The centrifuge tube was refilled with acetone (15 mL) and centrifuged to recover silica nanoparticles. The precipitation and redistribution process was repeated four times to wash the nanoparticles. Finally, the nanoparticles were dried under a gentle stream of nitrogen to obtain an orange powder (45 mg). Elemental Analysis of Azide-Coated Nanoparticles (4dark). The weight fraction of nitrogen in the “dark” nanoparticles is given by eq 129

Figure 1. Myelin glycolipids.

Model systems have been valuable in establishing the CCI between GalCer and SGalCer. In a study of the aggregation of various glycolipid-containing liposomes in the presence of various divalent cations, the highest amount of aggregation was observed between vesicles containing GalCer and vesicles containing SGalCer.49 This interaction was studied using fluorescent and spin label probes, as well as antiglycolipid antibodies, all of which indicated a preferential trans-bilayer interaction between the glycolipids.50 If the GalCer and SGalCer were embedded in PC/cholesterol bilayers, divalent cations were required for the CCI to occur.49 However, if bilayers of only GalCer and SGalCer were used, the interaction occurred in the absence of divalent cations.50 Ternary complexes of GalCer, SGalCer, and calcium have been detected using electrospray mass spectrometry.51 More recent studies have shown that the Gal•SGal CCI is involved in signal transduction in oligodendrocytes. Addition of GalCer and SGalCer-containing liposomes to oligodendrocytes resulted in redistribution of GalCer and proteins on the oligodendrocyte cell surface.52,55 Consistent with the hypoth-

x × 3 × MWN 4 3 πr 3

× d + x × MWcoating

= N% (1)

where r is the radius of the silica nanoparticles (17 nm as determined by TEM; see Supporting Information), d is the density of silica (2.0 × 10−21 g/nm3), MWN and MWcoating are the formula weights of nitrogen and the silane on the nanoparticle surface, respectively. x stands for the moles of azidosiloxane 2 on the surface of each nanoparticle. B



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μL). A control experiment was carried out with 4-dark that had been functionalized with propargyl alcohol (A = 0.001). The absorbance of galactose-coated nanoparticles is fitted to the calibration curve (Supporting Information) to calculate the concentration of carbohydrate on the surface of silica nanoparticles. CCI-Induced Aggregation of Carbohydrate Coated Silica Nanoparticles. 0.1 mg/mL solution of 3-sulfogalactose coated fluorescent silica nanoparticles and 0.1 mg/mL solution of different carbohydrate-coated dark silica nanoparticles were prepared in HEPES buffer (0.05 M HEPES buffer with 0.15 M NaCl, pH = 7.11). The 3-sulfogalactose-coated fluorescent silica nanoparticles solution (1 mL) and the other carbohydrate-coated dark silica nanoparticles solution (1 mL) were combined. After 30 min, samples for TEM analyses were prepared by drying solutions of the particles on amorphous carbon-coated copper grids. Measurement of CCI Using High-Binding Microtiter Plates. A methanolic solution of glycolipid 7a or 7b (50 μL, 0.1 M) was loaded in each well. The microtiter plate was placed in a fume hood, and the solvent was evaporated at room temperature in the hood; the wells were incubated with a 1% BSA in PBS buffer solution (120 μL, PBS buffer solution (pH = 7.1)) overnight. The wells were then incubated with fluorescent nanoparticles (100 μL, 1 mg/mL) in a 1% BSA in PBS buffer solution (pH = 7.1) for 1 h. The binding buffer solution was removed, and the wells were washed with 1% BSA in PBS buffer solution twice. Each wash consisted of three agitations of the solution using an automatic pipettor. The fluorescence was measured at λex 480 nm and λem 519 nm when buffer solution was added to the wells for the third time. Measurement of Binding of Carbohydrate-Coated Silica Nanoparticles to Cultured Oligodendrocytes and Myelin. Oligodendrocytes were cultured as described previously.55 The medium was replaced on day 8 in culture with phenol red-free medium (Invitrogen Canada, Inc.), and the live cells were treated with 40 μg/mL glycoparticles in medium for 1 h. They were then washed three times with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and stained with anti-MAG to visualize oligodendrocytes or antiglial fibrillary acidic protein (GFAP) to detect astrocytes, plus fluorescent Cy3-conjugated second antibodies, as described. For some experiments, cells were fixed first before addition of glycoparticles. If O1 Ab was added to block binding, it (1 μg per well) was added to live or fixed cells for 15 min, followed by washing three times. Glycoparticles were then added as above. Prepared slides were viewed in a Zeiss confocal laser scanning microscope with LSM 510 program. The green (FITC) and red (Cy3) data sets were acquired separately at 488 and 553 nm, respectively. Each optical section was 0.45 μm thick, and the sections showing the most intense staining are presented. A Reichert-Jung fluorescence microscope was used for initial viewing of the cells. The percent of MAG+ cells with a number of bound particles (sufficient to reveal the cell shape and different from random background particles) was determined by counting cells starting in one corner of the coverslip and proceeding through the coverslip until 300 cells were counted. This was usually one-half to two-thirds of the cells on the coverslip. CM-DiI-labeled myelin (10 μL containing 170 μg of protein) was combined with 20 μL (20 μg) FITC-Gal/SGal particles 5e or FITC−OH particles 5f. For blocking experiments, 10 μL

According to eq 1 {x[mol/particle] × 3 × 14.01[g/mol]} ⎧4 /⎨ π × 173[nm 3/particle] × 2.0 × 10−21[g/nm 3] ⎩3

}

+ x[mol/particle] × 457.71[g/mol]

(2)

= 2.84% x = 3.3 × 10−20[mol/particle]

The functional groups/particle is given by 3.3 × 10−20[mol/particle] × 6.02 × 1023[groups/mol] = 19866[groups/particle]

(3)

2

The functional groups/nm is given by 19866[groups/particle]/4π × 172[nm 2 /particle] = 5.5[groups/nm 2]

The mol/mg of linker on the surface of nanoparticles is calculated as follows: 2.84% = 0.68[μmol/mg] 3 × 14.01[g/mol]

(4)

General Reaction Protocol for Copper-Promoted Azide Alkyne Cycloadditions on 4 to Provide 5a−c. Azide-coated fluorescent Si NPs (10 mg, 6.8 μmol) were suspended in THF (2 mL) in a 3 mL microwave reactor vessel. An aqueous solution of propargyl pyranoside (34 μL, 1 M, 5 equiv) was added, followed by an aqueous solution of CuSO4 (34 μL, 0.1 M, 0.5 equiv) and an aqueous solution of sodium ascorbate (34 μL, 0.2 M, 1 equiv). The reactor vessel was put into the microwave reactor and heated at 70 °C for 10 min. After the reaction, methanol (10 mL) was added to precipitate the nanoparticles and the suspension was centrifuged for 5 min. The supernatant was discarded, and the precipitation and redispersion steps were repeated with 0.1 M EDTA solution (3×), nanopure water, and acetone. A gentle stream of nitrogen was used to dry the nanoparticles to provide an orange powder (11 mg). Synthesis of 3-Sulfogalactose-Coated Fluorescent Si NPs5d. Azide-coated fluorescent Si NPs (10 mg, 6.8 μmol) were dissolved in THF (2 mL) in a 3 mL microwave reactor vessel. A methanolic solution of sodium propargyl 3-sulfonatoβ-D-galactopyranoside (100 μL, 1 M, 5 equiv) was added. After that, CuI (19 mg, 0.1 mmol, 15 equiv) and diisopropylethylamine (20 μL, 0.1 mmol, 15 equiv) were added. The reactor vessel was put into the microwave reactor and heated at 70 °C for 3 h. The nanoparticles were precipitated, washed, and dried as above to provide an orange powder (12 mg). Determination of Carbohydrate Loading of 5a Using Phenol/Sulfuric Acid. Galactose-coated silica nanoparticles 5a (1 mg) were dissolved in nanopure water (2 mL). This solution (200 μL) was added to a glass vial. To this vial was added an aqueous solution of phenol (0.5 M, 200 μL), followed rapidly by concentrated sulfuric acid (1000 μL). After 30 min, the absorbance was measured at 490 nm (A = 0.479). Blank absorbance measurements contained an aqueous solution of nonfunctionalized silica nanoparticles (0.5 mg/mL 200 μL), phenol solution (200 μL), and concentrated sulfuric acid (1000 C



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Scheme 1. Synthesis of Carbohydrate-Functionalized Silica Nanoparticles and Glycolipids

Quantification of azide incorporation into the nanoparticles was carried out using elemental analysis for carbon, hydrogen, and nitrogen (Table 1). Bare nanoparticles lacking both fluorophore and azide contain a trace amount of nitrogen along with low amounts of carbon and hydrogen. The latter components are presumably derived from residual ethoxy groups within the silica matrix. In order to simplify interpretation of the elemental analysis data, azide-functionalized nanoparticles lacking the fluorescent dye were prepared. These “dark” analogues of 4 contain carbon, hydrogen, and nitrogen in ratios that reflect their composition in the silanol derived from the linker 2 (Table 1). The 2.84% nitrogen content in these nanoparticles is derived solely from the azide and translates to an azide loading of 0.68 μmol/mg and a density of 5.5 azides/nm2. Functionalization of the particles using alkyne-terminated glycosides was subsequently conducted. Reaction progress was monitored by following the loss of the azide peak in the IR spectrum. The reaction of 4 with propargyl β-D-galactopyranoside 6a to give 5a was carried out under microwave irradiation using 2.5 equiv of the alkyne, 50 mol % copper sulfate, and 100 mol % sodium ascorbate in a THF/water mixture. Under these conditions, complete conversion, as determined by IR, was achieved in 10 min at 70 °C. Purification was straightforward, as the nanoparticles could be recovered after several washing and precipitation cycles. TEM analysis of the nanoparticles before and after reaction showed that the shape and size distribution of the particles remained largely unchanged, indicating that the nanoparticles were stable under the reaction conditions. The azide-terminated nanoparticles were functionalized with propargyl β-D-glucopyranoside 6b and α-D-mannopyranoside 6c to provide particles 5b and 5c. The reaction of propargyl 3sulfo β-D-galactopyranoside 6d with the azide-terminated nanoparticles was carried out using copper(I) iodide as catalyst with complete conversion obtained under microwave irradiation at 70 °C for 3 h. Nanoparticles 5e, functionalized with both 6a and 6d in approximately equal ratios, were prepared by functionalization with the sulfo sugar 6d for 1.5 h followed by subsequent capping with 6a. Carbohydrate functionalization of the nanoparticles 5a−e was quantitated using elemental analysis (Table 1, entries 3−6). As before, these analyses

myelin was combined with a 4-fold excess of dark Gal/SGal particles (80 μL containing 80 μg particles) or 20 μL (20 μg) mouse monoclonal anti-GalCer O1 IgM antibody or 20 μL (76 μg) rabbit anti-GalCer IgG Fab for 1.5 h. The final volume was adjusted to 90 μL with distilled water, and then 20 μL (20 μg) FITC-Gal/SGal particles 5e were added. An aliquot (5.5 μL) of 200 mM CaCl2 solution was added to give a final concentration of 10 mM CaCl2. The samples were incubated for 2 h at room temperature. They were mounted on slides and examined using a confocal microscope.

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RESULTS AND DISCUSSION Nanoparticle Synthesis and Characterization. Fluorescein isothiocyanate (FITC) doped silica nanoparticles were synthesized following an adaptation of reported procedures (Scheme 1).56,57 The surface of these nanoparticles was subsequently coated with the azide-containing siloxane 2, which was synthesized from the commercially available chlorosiloxane 1. The diameter of the azide-modified nanoparticles (4), measured using TEM, was 54 ± 4 nm. The IR spectrum of these nanoparticles exhibited a strong peak at 2100 cm−1 corresponding to the azide antisymmetric stretch (Figure 2). The fluorescence spectrum of 4 exhibited the characteristic emission curve of fluorescein (Supporting Information) with a maximum at 514 nm (λex 480 nm).

Figure 2. IR spectra of (a) azide-functionalized particles 4, solid line; (b) fully functionalized particles 5a, dashed line. D



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Table 1. Elemental Analysis of Silica Nanoparticles

a

entry

nanoparticle (coating)

wt % C

wt % N

wt % H

1 2 3 4 5 6 7

bare silica 4 (N3) 5a (Gal) 5b (Glc) 5c (Man) 5d (SGal) 5e (SGal+Gala)

2.27 11.92 15.39 15.94 15.04 14.35 19.94

0.15 2.84 2.56 2.66 2.51 2.37 2.47

1.05 1.25 1.86 1.97 1.90 1.79 1.91

wt % S

mole ratio C:N

coverage μmol/mg

functional groups per nm2

1.81 0.95

4.9:1 7.0:1 7.0:1 7.0:1 7.0:1 7.0:1

0.68 0.61 0.63 0.60 0.56 0.59

5.5 5.6 5.9 5.6 5.6 5.7a

SGal:Gal = 2.9:2.8.

were carried out with the “dark” analogues of 5 lacking fluorescein in order to simplify data analysis. Functionalization of 4 with carbohydrates resulted in an increase in carbon and hydrogen weight percentages and a decrease in the nitrogen percentage. The surface coverage remains fairly constant for all three sugars, at approximately 0.6 μmol/mg. The lower molar loading value (0.68 vs 0.61 μmol/mg) reflects the increase in the molecular weight of the coating on the silica nanoparticles (42 for the azide vs 457 for the glycosyl triazoles). These correspond to 90 000 and 78 000 azide and carbohydrate groups per particle, respectively, translating to an 87% conversion based on elemental analysis. The presence and concentration of carbohydrates on the nanoparticles were also confirmed by treating the nanoparticles with phenol in sulfuric acid.58 This colorimetric assay indicated that the concentration of galactose on 5a is 0.58 μmol/mg, results that are consistent with those derived from elemental analysis. To provide additional confirmation of triazole formation, HF•pyridine was used to dissolve the silica nanoparticles, providing a triazole-containing glycoside, which was characterized using 1H NMR and mass spectrometry.59 Nanoparticle CCIs. To study the CCI of the carbohydratecoated nanoparticles, we examined their binding to glycolipidcoated microtiter plates, a glycan presentation that has been used previously for screening both CCI as well as carbohydrate−protein interactions.60,61 Glycolipids 7a,b containing a single hydrocarbon chain were prepared by the reaction of propargyl glycosides 6a,b with azidohexadecane. The lipids were subsequently immobilized in a 96-well polystyrene microtiter plate. The loading of the glycolipid in the wells was quantitated using phenol/sulfuric acid. Previous experiments with a similar glycolipid indicated that a hexadecyl chain was retained most effectively after multiple washing steps, and we find that this holds true with our glycosyl triazole lipids as well.61,62 We examined the binding of SGal-coated nanoparticles 5d to wells coated with galactolipid 7a. Variation of the incubation conditions identified a 60 min incubation of nanoparticles in PBS buffer at a 500 μg/mL concentration as being optimal for providing the maximal signal. We observed almost no difference in fluorescence signal when the binding was carried out in the presence or absence of 100 mM calcium ions, as observed for binding of pure GalCer to pure SGalCer.49 The CCI between the nanoparticles and the glycolipid is extremely specific for galactose and SGal, as seen in Table 2. When the wells are loaded with galactolipid, significant fluorescence is only observed when nanoparticles coated with SGal (5d) or a mixture of SGal and Gal (5e) are used. Only background levels of fluorescence are observed with the Gal and Glc nanoparticles 5a and 5b. A nanoparticle functionalized with propargyl alcohol (5f) was used as a control to evaluate

Table 2. Fluorescence Measurements of Nanoparticle Binding to Glycolipids in Multiwell Plate glycolipid coating in well nanoparticle (sugar coating) 5d (SGal) 5e (Gal + SGal) 5a (Gal) 5b (Glc) 5fa (OH)

7a (Gal) 3163 2780 498 465 465

± ± ± ± ±

80 70 59 64 61

7b (Glc) 467 523 499 434 550

± ± ± ± ±

58 65 47 62 50

a

5f was prepared by capping 4 with propargyl alcohol using the standard click conditions.

the role of nonspecific binding of the nanoparticles. In contrast, when the glucolipid 7b was immobilized in the wells, no binding was observed with any of the nanoparticles. These results demonstrate that the carbohydrate coated nanoparticles can be used as a platform for studying the CCI between SGal and Gal. Additional corroboration of the ability of the glyconanoparticles to engage in CCI was provided by TEM images of various nanoparticle combinations. Mixtures of fluorescent and dark nanoparticles could be distinguished in the TEM by virtue of their differences in size and contrast. The dye-loaded nanoparticles appear darker than nanoparticles lacking the dye and the former are larger in diameter. Examination of TEM images of fluorescent SGal nanoparticles mixed with nonfluorescent Gal nanoparticles shows large aggregates containing both types of nanoparticles within the aggregate (Figure 3A). In contrast, when a combination of the fluorescent SGal nanoparticles 5d mixed with nonfluorescent Glc nanoparticles 5b is examined, very few mixed aggregates are seen, and each type of particle is most likely to be found in an aggregate of like particles (Figure 3B).

Figure 3. TEM images of (A) a mixture of dark Gal nanoparticles (0.1 mg/mL) and fluorescent SGal nanoparticles (0.1 mg/mL); (B) a mixture of dark Glc nanoparticles (0.1 mg/mL) and fluorescent SGal nanoparticles (0.1 mg/mL). E



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We have previously shown that nonfluorescent “dark” Gal, SGal, and Gal/SGal coated particles induce the same redistribution of cell surface GalCer that is seen with liposomes and Gal-BSA.54 However, those studies did not detect glycoconjugate binding directly; instead, carbohydrate binding was inferred from the cellular effects of the glycoconjugates, inhibition using anti-GalCer antibodies, and additional experiment using glycolipid biosynthesis inhibitors.53 Using fluorescent silica nanoparticles, we sought to directly visualize the interaction of Gal and SGal-containing nanoparticles with oligodendrocytes. Oligodendrocytes were isolated from rat pup spinal cords and cultured and were identified by staining with an antibody to myelin-associated glycoprotein (MAG; red/ yellow stains in Figure 4A−D). Particle binding to oligodendrocytes was determined directly using the fluorescein emission (green/yellow stains in Figure 4). Both the Gal/SGal and Gal particles 5e and 5a bound to live oligodendrocytes (Figure 4A,B), and weak binding of the SGal particles 5d was also observed (SI). Glucose and mannose functionalized particles 5b and 5c did not bind significantly to oligodendrocytes, and 5e did not bind to astrocytes present in the culture, which lack GalCer and SGalCer (SI). If cell surface GalCer is indeed the target for SGal on the Gal/SGal particles, the binding should be reduced if a competing ligand is present. Indeed, incubation of oligodendrocytes with the O1 anti-GalCer monoclonal antibody for 15 min before addition of 5e greatly reduced binding of the particles to the oligodendrocytes but had no effect on the binding of 5a (Figure 4C,D and Table 3). The antibody completely prevented the weak binding of 5d (SI) These results are consistent with the conclusions that (a) the ligand for SGal particles is galactosylceramide (GalCer) in the oligodendrocyte membranes and (b) the ligand for Gal particles in the oligodendrocyte membrane is SGal, which would not be blocked by anti-GalCer.

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Figure 4. (A−D) Fluorescence microscope images of nanoparticles (green) bound to live oligodendrocytes followed by fixation and staining with anti-MAG Ab (red); yellow indicates overlap. (A) Gal/ SGal nanoparticles 5e; (B) Gal nanoparticles 5a; (C) *Cells were incubated with Anti GalCer O1 Ab added for 15 min prior to binding of 5e; (D) Cells were incubated with anti-GalCer O1 Ab added for 15 min prior to binding of 5a. (E,F) Fluorescence microscope images of nanoparticles (green) bound to myelin fragments labeled with CD-DiI (red). (E) Gal/SGal nanoparticles 5e; (F) 76 μg anti-GalCer IgG Fab fragments followed by 5e. Scale bars = 20 μm. Representative pictures are shown. [*Anti-GalCer Ab added to live cells for 15 min causes some damage to the cells, resulting in a number of small MAG+ condensed cells. One large cell remaining has greatly reduced MAG staining. Results for binding to fixed cells are provided in the SI.]

CONCLUSIONS In conclusion, we have developed a multivalent platform for presenting carbohydrates and studying their CCI. Fluorescent silica nanoparticles were efficiently functionalized with carbohydrates via a copper-promoted click reaction. Attaching the azide coupling partner on the nanoparticle facilitates reaction monitoring by tracking the disappearance of the diagnostic azide IR band. High levels of functionalization can be obtained and the functionalization conditions are mild and general, which should permit the attachment of a wide variety of additional ligands on nanoparticles. Particles coated with SGal as well as a mixture of Gal and SGal adhered to multiwell plates coated with a galactolipid and preferentially formed mixed aggregates with their binding partner on a TEM grid. These experiments indicate that the Gal•SGal CCI is also extremely specific for those two sugars, as glucose-coated nanoparticles did not interact with SGal. Additionally, these findings indicate that the Gal•SGal CCI does not require the sphingolipid chain, and that monosaccharides are necessary and sufficient for the CCI, similar to other glycolipid CCIs that have been studied using model systems. The use of a fluorescent multivalent glycoconjugate allowed us to probe CCI both using model systems, as well as in a more complex and heterogeneous cellular environment. Particle binding to oligodendrocytes was examined using fluorescence microscopy, and enabled the direct visualization of the binding

Table 3. FITC-Containing Nanoparticle Binding to Oligodendrocytes and in Presence and Absence of antiGalCer O1 Antibodya % of MAG+ oligodendrocytes with bound particles nanoparticles

no Ab

with O1 Ab

5e 5a

74 92.1

29.4 95.5

a

The Gal/SGal nanoparticles 5e also bound to purified myelin fragments (Figure 4E). The binding was inhibited by an excess of the “dark” nonfluorescent version of 5e (SI) and also by a monovalent anti-GalCer IgG Fab fragment (Figure 4F).

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of Gal and SGal glycoconjugates to these cells. These results provide a new tool for studying the Gal•SGal and other CCIs. The fluorescence readout allows for the rapid evaluation of the effect of carbohydrate structural modifications to CCI and can be used to screen for compounds that can inhibit the interaction, particularly in a cellular context.

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ASSOCIATED CONTENT

* Supporting Information S

Additional experimental procedures, microscopy images, and characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Science Foundation (A.B.), the Multiple Sclerosis Society of Canada (J.M.B.), and in part by a Research Seed Fund from Brown University (A.B.).

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REFERENCES

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Carbohydrate-Coated Fluorescent Silica Nanoparticles as Probes for

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