Analysis of CarbohydrateâCarbohydrate Interactions Using Sugar

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Analysis of carbohydrate-carbohydrate interactions using sugar-functionalized silicon nanoparticles for cell imaging Chian-Hui Lai, Julia Hütter, Chien-Wei Hsu, Hidenori Tanaka, Silvia VarelaAramburu, Luisa De Cola, Bernd Lepenies, and Peter H Seeberger Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.5b04984 • Publication Date (Web): 13 Dec 2015 Downloaded from http://pubs.acs.org on December 16, 2015

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Analysis of carbohydrate-carbohydrate interactions using sugar-functionalized silicon nanoparticles for cell imaging Chian-Hui Lai, 1, ‡ Julia Hütter, 1,2, ‡ Chien-Wei Hsu,3 Hidenori Tanaka,1Silvia Varela-Aramburu, 1,2 Luisa De Cola,3 Bernd Lepenies, 1, 2,* Peter H. Seeberger 1, 2, *

1

Max Planck Institute of Colloids and Interfaces, Department of Biomolecular

Systems, Potsdam, Germany

2

Freie Universität Berlin, Institute of Chemistry and Biochemistry, Department of

Biology, Chemistry and Pharmacy, Berlin, Germany

3

Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université Strasbourg, 8

allée Gaspard Monge, 67083 Strasbourg, France.

1 ACS Paragon Plus Environment

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ABSTRACT Protein-carbohydrate binding depends on multivalent ligand display that is even more important for low affinity carbohydrate-carbohydrate interactions. Detection and analysis of these low affinity multivalent binding events are technically challenging. We describe the synthesis of dual-fluorescent sugar-capped silicon nanoparticles that proved to be an attractive tool for the analysis of low affinity interactions. These ultra-small NPs with sizes of around 4 nm can be used for

NMR quantification of

coupled sugars. The silicon nanoparticles are employed to measure the interaction between the cancer-associated glycosphingolipids GM3 and Gg3 and the associated kD value by surface plasmon resonance experiments. Cell binding studies, to investigate the biological relevance of these carbohydrate-carbohydrate interactions, also benefit from these fluorescent sugar-capped nanoparticles.

KEYWORDS: Carbohydrate-carbohydrate interactions, silicon nanoparticles, low-affinity binding, surface plasmon resonance, real-time imaging


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Glycans are prominently exposed on the surface of living cells and are critically involved in cell-cell interactions and regulate important biological functions such as cell growth and cell differentiation.1 Carbohydrate-protein interactions are typically very weak but are key to biological processes that require temporary adhesion such as during cell adhesion or leukocyte recruitment.1 Multivalent presentation compensates for the low affinity of a single binding event.2 The oligosaccharide portion of cell surface glycoproteins and glycolipids interact with carbohydrate-binding proteins or other oligosaccharide chains by glycoside cluster effects.3 Carbohydrate-protein interactions

are

characterized

by

low

affinity

binding,

while

the

carbohydrate-carbohydrate interactions (CCIs) between glycosphingolipids (GSL) are ultra-low affinity, a feature that is advantageous during the first phase of some cell adhesion

events.4,5

Plasma

membrane

microdomains

are

enriched

in

glycosphingolipids that participate in multivalent CCIs as the initial step of cell recognition.6 Lex/Lex interactions for example are involved in morula compaction during mouse embryogenesis.7 Sialosyllactosylceramide (GM3) present on melanoma cells engage gangliotriaosylceramide (Gg3) on lymphoma cells during the initial adhesion of melanoma cells to the endothelium thus contributing to tumor metastasis.8 While a detailed picture of carbohydrate-protein interactions has evolved,9,10 relatively little is known about low affinity CCIs and their biological relevance. 3 ACS Paragon Plus Environment

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Various CCI model systems have been proposed,11-19 but to date only limited data is available due to the difficulties associated with detecting these ultra-weak interactions. A reliable technique to quantitatively analyze CCIs is urgently needed. Adhesion forces of Lex/Lex interactions were determined using atomic force microscopy measurements on self-assembled monolayers.11 Glycol-coated gold nanoparticles that mimic the multivalent carbohydrate presentation on cells were employed for TEM analysis12 and surface plasmon resonance (SPR) measurements.13 Glyco-monomers and glycol-dimers were subjected to affinity analysis by NMR.14 GM3/lactose

interactions

between

glycolipid

Langmuir

monolayers

and

glycol-micelles15 or glycol-dendrimers were studied.16 Glycan-coated silica NPs were applied to detect the galactose and 3-sulfogalactose interplay.17 The GM3/Gg3 pair was quantitated by SPR using different GSLs as well as Gg3-carrying-polymers.18,19 In all cases multivalent glycan presentation was key to studying CCIs and carbohydrate-functionalized NPs proved attractive tools.20-27 The preparation of well-characterized sugar-capped NPs for biophysical and biological studies involving low-affinity CCIs has been very challenging.

We created structurally-defined and fluorescently labeled multifunctional carbohydrate-capped NPs to study ultra-weak CCIs by SPR and cell imaging. Silicon nanoparticles (SiNPs) were employed due to their ultra-small size, inherent 4 ACS Paragon Plus Environment

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luminescent properties, easy covalent surface functionalization and negligible cytotoxicity.28,29

An

additional

dye

(ATTO-647N)

was

added

to

create

dual-fluorescent SiNPs. Multi-fluorescent NPs, when compared to single-dye NPs, provide additional information and prevent false positives.30,31 The relatively well understood GM3-Gg3 interaction was selected as model system. Using the well-characterized dual-fluorescent sugar-capped SiNPs, we performed SPR measurements and analyzed the biological relevance of this interaction by using the B16F10 melanoma cell line that displays high levels of GM3.32 A multivalent scaffold was created by coupling the carbohydrate ligands and the additional dye to silicon nanoparticles (sugar-dye@SiNPs) (Scheme 1). Three different sugar-dye@SiNPs termed Glc-dye@SiNP, Lac-dye@SiNP, and Gg3-dye@SiNP, were synthesized, with the first two serving as controls. The sugar-dye@SiNPs were fully characterized (see below) and used for quantitative SPR analysis of the GM3/Gg3 interaction as well as for cell binding studies.


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Scheme 1. Synthesis of the different sugar-dye@SiNPs: Glc-dye@SiNPs, Lac-dye@SiNPs and Gg3-dye@SiNPs.

The carboxylic acid functionalized SiNPs (COOH@SiNPs) were synthesized and modified

according

to

literature

procedures

(Scheme

1

and

supporting

information).33,34 X-ray photoelectron spectroscopy (XPS) analysis confirmed the elemental compositions (Figure S1 and Table S1). According to TEM (Figure 1a), 4 ± 1 nm COOH@SiNPs are consistent with the size determined by analytical ultracentrifugation (AUC) measurements (Figure S2). The average molecular weight of the COOH@SiNPs was further determined at 10.5 kDa according to AUC measurements.35 Acid-base titration revealed that 1.17 µmol carboxyl acid groups are present per mg of COOH@SiNP (Figure S3). Considering the particle size determined by TEM and the average molecular weight measured by AUC, 15 carboxylic acid groups should be present on a single COOH@SiNP particle on average. An amino-functionalized ATTO-647N dye was conjugated to the COOH@SiNP in the presence of the coupling reagents EDC and HOBt in DMF and 6 ACS Paragon Plus Environment

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NEt3 at room temperature for 24 hours to obtain green COOH-dye@SiNPs (Scheme 1, Figure S4). Analysis of COOH@SiNPs and COOH-dye@SiNPs by dynamic light scattering (DLS) and UV absorption revealed equal distribution and successful dye coupling (Figure S4). Amino-functionalized carbohydrate ligands, glucose 1, lactose 2, and Gg3 oligosaccharide 3 were synthesized using modifications of reported procedures (for 1H-NMR spectra of these compounds, see Figures S5, S6 and S7).25,36,37 Following amide bond formation of COOH-dye@SiNPs and the carbohydrate ligands 1, 2 and 3 for 48 hours, the remaining carboxylic acid groups on SiNPs were capped with ethanolamine to afford sugar-dye@SiNPs (Glc-dye@SiNP, Lac-dye@SiNP and Gg3-dye@SiNP). After purification by size-exclusion chromatography and dialysis, all SiNPs formulations were characterized regarding the zeta potential of their surface charge (Figure 1b), their hydrodynamic diameter by DLS (Figure S8), and individual functional groups on the particles by infrared spectroscopy (Figure S9). The zeta potential measurements provided valuable information on the success of sugar placement since the negative charge was markedly reduced after coupling (Figure 1b). The surface chemical bonding on sugar-dye@SiNPs was further characterized by NMR spectroscopy (Figure 1c, Figure S10 and Figure S11). SiNPs have the great advantage of being usable in NMR for quantification of the coupled sugar moieties. The number of sugars on 7 ACS Paragon Plus Environment

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Glc-dye@SiNPs, Lac-dye@SiNPs and Gg3-dye@SiNPs were determined at 12.2, 12.1 and 8.3 per particle by 1H-NMR analysis. This corresponds to coupling yields of around 82%, 81% and 56% based on carboxyl group density COOH@SiNPs (Table S2). The theoretical molecular weight of Glc-dye@SiNPs, Lac-dye@SiNPs, and Gg3-dye@SiNPs was calculated at13.7, 15.7, and 15.7 kDa, respectively. Individual anomeric sugar carbons of different sugar-dye@SiNPs were identified by 13C-NMR (Figure S11). Photo-luminescence excitation spectra of COOH-dye@SiNPs and sugar-dye@SiNPs revealed similar profiles with expected fluorescence bands around 450 and 660 nm (Figure S12). Analysis and comparison of XPS data for the COOH-dye@SiNPs and sugar-dye@SiNPs (Figure S13 to S16 and Table S3 to S6) revealed that all samples contained Si, C, and O and maintained similar physical properties as COOH@SiNPs (Figure S1 and Table S1); the presence of nitrogen derives stems from the amide formed with the dye or the sugar ligands. TEM analysis of the sugar-dye@SiNPs excluded particle aggregation (Figure S17).


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Figure 1. (a) Representative TEM image of COOH@SiNPs indicates particle sizes of around 4 nm. (b) Zeta-potential of the various particle formulations (n = 6). (c) 1

H-NMR spectra of the different sugar-dye@SiNPs.

Multivalent carbohydrate presentation on SiNPs allows for analysis of low-affinity carbohydrate-protein and carbohydrate-carbohydrate interactions. To perform affinity analyses, sugar-dye@SiNPs were used for SPR measurements. First, the sugar-dye@SiNPs were used to determine a model protein-carbohydrate interaction, between the asialoglycoprotein receptor (ASGPR) and terminal galactose (Gal) and N-acetylgalactosamine (GalNAc) residues to verify their utility for SPR studies.


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Recombinant human ASGPR was immobilized on a SPR chip and its binding to the sugar-dye@SiNPs was analyzed. Indeed, specific binding of the Gg3-dye@SiNPs to ASGPR was detected (Figure S18). The interaction was markedly stronger than binding to Glc-dye@SiNPs or Lac-dye@SiNPs, as is expected based on the higher affinity of the ASGPR to terminal GalNAc than for Gal.38 Since Gg3 includes a terminal GalNAc, ASGPR is expected to bind Gg3 stronger than Lac. Clearly, sugar-dye@SiNPs are very useful for SPR-based binding studies.

Next, Gg3-dye@SiNPs were used for SPR analysis of the GM3/Gg3 interaction. To

this

end,

GM3-functionalized

and

biotinylated

poly[N-(2-hydroxyethyl)acrylamide] polymers (GM3-biotin-PAA) were immobilized on a SPR sensor chip to mimic the multivalent glycan presentation on a cell surface while the sugar-dye@SiNPs were used as analyte. As a specificity control, Lac-biotin-PAA was immobilized on the reference flow cell. Indeed, immobilized GM3 was bound specifically to Gg3-dye@SiNPs but was not bound to Glc-dye@SiNPs or Lac-dye@SiNPs (Figure 2, A-C). A specific interaction corresponds to the signal obtained by subtraction of the response units detected in the reference flow cell. Steady-state affinity analysis for binding of Gg3-dye@SiNPs to immobilized GM3 resulted in an apparent kD value of around 5.5 x 10-7 M using a theoretical molecular weight of the Gg3-dye@SiNPs of 15.7 kDa. This kD value is 10 ACS Paragon Plus Environment

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comparable to the apparent binding constant kA of 2.5 x 106 M-1 (kD = 1/kA) reported earlier for the Gg3/GM3 interaction based on SPR studies using polystyrene glycoconjugates.18,19

Figure 2. Multivalent GM3/Gg3 interactions analyzed by SPR measurements. The GM3-biotin-PAA polymer was immobilized on a streptavidin-coated SA sensor chip. 11 ACS Paragon Plus Environment

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As control, the Lac-biotin-PAA polymer was immobilized on the reference flow cell. Various concentrations (20-80 µg/mL) of (A) Glc-dye@SiNPs, (B) Lac-dye@SiNPs, or (C) Gg3-dye@SiNPs were flowed over the chip surface as analytes to detect binding to the immobilized GM3. The SPR sensorgrams were generated by subtraction of the reference flow cell as well as of the signals obtained by injection of the running buffer alone. Specific binding was only observed for Gg3-dye@SiNPs.

The encouraging SPR measurements were followed up by a cell-based assay to investigate binding of Gg3-functionalized SiNPs to GM3. To this end, the uptake of the sugar-dye@SiNPs by B16F10 cells, a GM3-overexpressing melanoma cell line, was determined by flow cytometry. The GM95 cell line served as control since this mutant B16F10 cell line does not express the GM3 ganglioside.2 The uptake of Gg3-dye@SiNPs by B16F10 cells was markedly higher than that of Glc-dye@SiNPs and

Lac-dye@SiNPs

(Figure

S19).

However,

unspecific

uptake

of

the

sugar-dye@SiNPs by GM95 cells was also observed (data not shown). Therefore, cell uptake of the sugar-dye@SiNPs was subsequently further analyzed by confocal fluorescence microscopy to confirm the specificity of the sugar-dye@SiNP/cell interactions and to determine the utility of the sugar-dye@SiNPs for cell imaging applications. B16F10 and GM95 cells were seeded onto sterile glass coverslips and were incubated with 20 µg/mL of the different sugar-dye@SiNPs at 37 °C for 2 12 ACS Paragon Plus Environment

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hours,

respectively.

Two

fluorescence

signals

were

detected

for

the

sugar-dye@SiNPs: one, the blue color, was associated with the inherent fluorescence of SiNPs and the second, purple fluorescence was derived from the additional dye on the surface of the Si-NPs. These studies revealed that Gg3-dye@SiNPs were indeed internalized into the GM3-expressing B16F10 melanoma cells (Figure 3 (a) and (e)). Uptake of Gg3-dye@SiNPs into the B16F10 cells was confirmed by Z-stack analysis (Figure S21-1 to S21-12). In contrast, Glc-dye@SiNPs and Lac-dye@SiNPs were not taken up (Figure S22 to S23). Differential interference contrast (DIC) microscopy confirmed the normal cell morphology of B16F10 cells (Figure 3b). In addition, early endosomes were stained with an anti-EEA1 (early endosome antigen 1) antibody (Figure 3c) and Alexa 555-labeled phalloidin was further added to the cells to visualize the actin cytoskeleton (Figure 3d). The overlay of different fluorescent signals revealed that the Gg3-dye@SiNPs were taken up by the B16F10 cells but did not co-localize with early endosomes (Figure 3f). Moreover, cells exhibited normal cell morphology and actin cytoskeleton staining upon incubation with the sugar-dye@SiNPs. Z-stack analysis of GM3 non-expressing GM95 cells showed only marginal uptake of the Gg3-dye@SiNPs and no uptake of the Glc-dye@SiNPs and Lac-dye@SiNPs (Figure S25 to S27). Thus, confocal microscopy studies


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revealed specific uptake of the Gg3-dye@SiNPs by the GM3-expressing B16F10 cells.

Figure 3. Representative confocal fluorescence microscopy images showing the uptake of Gg3-dye@SiNPs by B16F10 cells after incubation with 20 µg/mL of SiNPs at 37°C for 2 h. (a) SiNPs were detected by their inherent fluorescence (blue color). (b) Normal B16F10 cell morphology was shown by differential interference contrast microscopy. (c) Early endosomes were stained with an anti-EEA1 antibody (green color). (d) The actin cytoskeleton was stained by incubation with Alexa 555-labeled phalloidin (red color). (e) SiNPs were further detected by the coupled ATTO-647N dye (purple color). (f) An overlay of the images shown in (a to e) with phase contrast. Scale bar: 20 µm.

Conclusion


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We demonstrate that carbohydrate-functionalized silicon nanoparticles are a multivalent

platform

to

study

ultra-low

affinity

interactions

such

as

carbohydrate-carbohydrate binding events. Three different sugar-capped SiNPs (Glc-dye@SiNP, Lac-dye@SiNP, and Gg3-dye@SiNP) were synthesized and extensively characterized. These sugar-capped SiNPs were subsequently used to determine the low-affinity interaction of the two glycosphingolipids GM3 and Gg3. Specific binding of Gg3-dye@SiNPs to immobilized GM3-biotin-PAA was detected and affinity analysis was performed by SPR confirming the existence of this carbohydrate/carbohydrate interaction. These studies further demonstrate that the sugar moieties of the two glycosphingolipids play a crucial role in their interaction since these experiments are only based on the sugar parts of GM3 and Gg3 excluding their lipid moieties. Importantly, sugar-dye@SiNPs are valuable tools for cell imaging as demonstrated by confocal fluorescence microscopy analysis of the uptake of Gg3-dye@SiNPs into GM3-expressing B16F10 melanoma cells. Thus, we here show that ultra-small SiNPs can be easily prepared as multivalent sugar-capped scaffolds for analysis of low-affinity carbohydrate-carbohydrate interactions in biophysical studies such as SPR as well as by cell imaging studies.

ASSOCIATED CONTENT


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Supporting Information

Details on synthesis and characterization of COOH@SiNPs; synthesis and characterization of sugar-dye@SiNPs; experimental details for the biological studies; SPR analysis; flow cytometry; Z-stack images of confocal microscopy. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors * Bernd Lepenies, E-mail: [email protected]

*Peter H. Seeberger, E-mail: [email protected]

Present Addresses (C.-H. L.) Genomics Research Center, Academia Sinica, Taipei, Taiwan

(J. H.) Section for Virology, National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark

(H. T.) Oceanography Section, Science Research Center, Kochi University, Kochi, Japan

(B. L.) University of Veterinary Medicine Hannover, Research Center for Emerging Infections and Zoonoses, Hannover, Germany 16 ACS Paragon Plus Environment

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Author Contributions ‡ C.-H. L. and J. H. contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the Max-Planck Society for generous financial support. Funding by the SFB765 (to P.H.S and B.L.) and the Helmholtz Virtual Institute NanoTracking Agreement Number VH-VI-421 (to C.-W.H. and L.D.C.), are also gratefully acknowledged. The authors wish to acknowledge the RIKEN BioResource Centre as the source of the GM95 cells through the National Bio-Resource Project of the MEXT, Japan. We thank Uwe Vogel for assistance with the preparation of confocal fluorescence images and Dr. Magdalena Eriksson for constructive discussions.


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(23) Kennedy, D. C.; Grunstein, D.; Lai, C. H.; Seeberger, P. H. Chem.- Eur. J. 2013, 19, 3794. (24) Chang, T.-C.; Lai, C.-H.; Chien, C.-W.; Liang, C.-F.; Adak, A. K.; Chuang, Y.-J.; Chen, Y.-J.; Lin, C.-C. Bioconjugate Chem. 2013, 24, 1895. (25) Lai, C.-H.; Lin, C.-Y.; Wu, H.-T.; Chan, H.-S.; Chuang, Y.-J.; Chen, C.-T.; Lin, C.-C. Adv. Funct. Mater. 2010, 20, 3948. (26) Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger, P. H. J. Am. Chem. Soc. 2009, 131, 2110. (27) Marradi, M.; Chiodo, F.; Garcia, I.; Penades, S. Chem. Soc. Rev. 2013, 42, 4728. (28) O’Farrell, N.; Houlton, A.; Horrocks, B. R. Int. J Nanomedicine 2006, 1, 451. (29) Rosso-Vasic, M.; Spruijt, E.; van Lagen, B.; De Cola, L.; Zuilhof, H. Small 2008, 4, 1835. (30) Wang, L.; Yang, C.; Tan, W. Nano Lett. 2005, 5, 37. (31) Gorris, H. H.; Ali, R.; Saleh, S. M.; Wolfbeis, O. S. Adv. Mater. 2011, 23, 1652. (32) Azuma, Y.; Ishikawa, Y.; Kawai, S. Clin. Cancer Res. 2007, 13, 4029. (33) Liu, Q.; Kauzlarich, S. M. Mater. Sci. Eng., B 2002, B96, 72. (34) Hsu, C.-W.; Septiadi, D.; Lai, C.-H.; Chen, P.; Seeberger, P.H.; De Cola, L. 2015 submitted for publication. (35) Carney, R. P.; Kim, J. Y.; Qian, H.; Jin, R.; Mehenni, H.; Stellacci, F.; Bakr, O. M. Nat. Commun. 2011, 2, 335. (36) Huang, L.-D.; Adak, A. K.; Yu, C.-C.; Hsiao, W.-C.; Lin, H.-J.; Chen, M.-L.; Lin, C.-C. Chem.- Eur. J. 2015, 21, 3956. (37) Esposito, D., PhD thesis, ETH Zurich, 2011, p169. (38) Lee, Y. C.; Lee, R. T.; Ernst, B.; Hart, G. W.; Sinaý, P. In Carbohydrates in Chemistry and Biology; Wiley-VCH Verlag GmbH: 2008, p 549.


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