Quantitative analysis of multivalent interactions of carbohydrate- encapsulated gold nanoparticles with concanavalin A

Article abstract of DOI:10.1039/b308995a

Multivalent interactions between carbohydrate-encapsulated gold nanoparticles and Con A are found with high affinity and specificity.

Full text of DOI:10.1039/b308995a

Quantitative analysis of multivalent interactions of
carbohydrate-encapsulated gold nanoparticles with concanavalin A†
Chun-Cheng Lin,*a Yi-Chun Yeh,b Chan-Yi Yang,b Gee-Fong Chen,b Yi-Chen Chen,b Yi-Chun Wu*c
and Chia-Chun Chen*b
a Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan
b Department of Chemistry, National Taiwan Normal University and Institute of Atomic and Molecular
Sciences, Academia Sinica, Taipei 116, Taiwan. E-mail: cjchen@cc.ntnu.edu.tw; Fax: 886-289316363;
Tel: 866-289316151
c
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan
Received (in Cambridge, UK) 31st July 2003, Accepted 14th October 2003
First published as an Advance Article on the web 28th October 2003
Multivalent interactions between carbohydrate-encapsu-
lated gold nanoparticles and Con A are found with high
affinity and specificity.
effective inhibitors of protein–carbohydrate interactions in
biological system.
Various carbohydrate-AuNP as described in Fig. 1 were
synthesized following similar methods as previously reported.13
The detailed procedures of synthesis and characterization of
carbohydrate-AuNP are described in ESI.† In brief, nearly
mono-dispersed gold nanoparticles with average diameters of 6
or 20 nm were prepared, and their sizes were confirmed using
transmission electron microscopy.14 Various carbohydrate
ligands with different linker lengths were synthesized with an
S–H group at one side, which was then linked to the
nanoparticle through the formation of a strong thiol bond. The
amount of carbohydrates attached on each gold nanoparticle
was quantitatively determined by H2SO4/phenol assay and
elemental analysis.15
Multivalent interactions between cell surface receptors and
carbohydrates have been discovered in a number of biological
processes including fertilization, proliferation, viral/bacterial
infection and the inflammatory response.1 Because of the
simultaneous binding of multiple ligands on one biological
entity to multiple receptors on another, the multivalent
interactions are often with high binding affinity and specificity.
A number of diverse scaffolds have been generated for
multivalent carbohydrate ligand presentation.2 Low-molecular
weight displays,3 copolymers,4 dendrimers,5 nanoparticles,6
and liposomes,7 for examples, have been employed as powerful
carriers to present carbohydrate ligands in polydisperse, linear
or globular architectures. In particular, LewisX (LeX, a
trisaccharide antigen) encapsulated on gold nanoparticles,6
mimicking glycosphingolipid clusters, exhibited significantly
enhanced affinity and selectivity as compared with LeX
monomers. However, the multivalent binding between carbohy-
drate-based nanoparticles and lectins has not been demonstrated
thus far.
Concanavalin A (Con A), a plant lectin, can form a tetramer
under appropriate conditions and bind specifically to manno-
and glucopyranosides.8 The binding between Con A and
carbohydrates has long been recognized as an excellent system
to study multivalent effects. Multivalent interactions have been
studied using various techniques such as electron microscopy,9
calorimetry,10 X-ray crystallography11 and surface plasmon
resonance (SPR).12 These studies have provided information on
the Con A and saccharide binding model and interaction
kinetics, which are useful for the development of potent
inhibitors and biological effectors.
To assess the binding affinity of a- -methyl-mannopyranno-
D
side (aMeMan) and various carbohydrate-AuNP for Con A, we
utilized the SPR competition binding assay based on the
previous report12,16 with some modifications. A self-assembled
monolayer composed of 20% mannopyranoside ligand and 80%
thiobutanol mixture was generated on a J1 biosensor chip (Fig.
1).17 The affinity of Con A for this chip was determined by
titration with series of Con A concentrations to generate
multiple SPR response curves. Using the rectangular hyperbolic
equation18 the association constant Ka of Con A for this chip
was obtained, and the value was 7.95 3 106 M21. In
competition assays, inhibition curves were generated by
measuring the binding responses for 0.5 mM Con A tetramer in
Recently, we have demonstrated that mannose-encapsulated
gold nanoparticles (m-AuNP) can be used as a probe to target
specific proteins in living bacteria.13 In this paper, we explore
the multivalent interactions between Con A and mannose-,
glucose- and galactose-encapsulated gold nanoparticles (abbre-
viated as m-AuNP, g-AuNP and t-AuNP, respectively, see Fig.
1). The SPR technique is applied to quantitatively analyze the
interactions. We found that the binding of m-AuNP to Con A
exhibited a strong multivalent effect and that the binding
specificity of Con A for the multivalent carbohydrate-encapsu-
lated gold nanoparticles (carbohydrate-AuNP) was similar to
that of the monovalent counterparts. We also show that the
affinity of m-AuNP for Con A can be adjusted by altering the
nanoparticle size or sugar moiety. Our results demonstrate that
nanoparticles can be excellent multivalent carbohydrate carriers
for lectins and that carbohydrate-AuNP has great potential as
Fig. 1 (a) Schematic illustration of the interactions of carbohydrate-AuNP
and Con A on the biosensor chip used in the competition assays. (b) Five
different carbohydrates (sugar-R) encapsulated on two different sizes of
gold nanoparticles are summarized. Specifically, gluco- and galactopyrano-
side are encapsulated on nanoparticles of 6 nm and abbreviated as 6-g-
AuNP and 6-t-AuNP, respectively. Mannopyranoside clustered on nano-
particles of 6 nm and 20 nm in diameter are abbreviated as 6-m-AuNP and
20-m-AuNP, respectively. Mannopyranoside with short or long linker
lengths are encapsulated on 20 nm nanoparticles to form s-20-m-AuNP and
l-20-m-AuNP, respectively.
† Electronic supplementary information (ESI) available: detailed experi-
mental procedures, SPR response curves and compound characterization
data. See http://www.rsc.org/suppdata/cc/b3/b308995a/
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CHEM. COMMUN., 2003, 2920–2921
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the presence of different concentrations of aMeMan or
carbohydrate-AuNP as competitive inhibitors. Fig. 2 presents a
set of SPR response curves for Con A in the presence of 6-m-
AuNP. From the inhibition curves of each carbohydrate-AuNP,
its inhibition constant (Ki) is obtained using the equations
derived by Attie et al.19 (Table 1). To compare the inhibition
potencies of the individual mannose ligand on three different m-
AuNP (6-m-AuNP, s-20-m-AuNP or l-20-m-AuNP) with re-
spect to monovalent aMeMan, we calculated the relative
inhibition potency (RIP).20
The RIP values for the mannose ligands of three m-AuNP are
from 11 to 128 (Table 1), indicating that the multivalent
mannose ligands of these m-AuNP have one to two orders
higher affinities to Con A than monovalent mannose ligands. In
addition, all three m-AuNP exhibited a stronger inhibition effect
than 6-g-AuNP and 6-t-AuNP. 6-t-AuNP displayed no detect-
able inhibition effect. This is consistent with the previous
studies that Con A binds to mannose better than glucose but
does not bind to galactose.21,12 Therefore, no switch of Con A
specificity for carbohydrates clustered on nanoparticles was
observed in our system. Taken together, our results demonstrate
that clustering of carbohydrate ligands on a nanoparticle
significantly enhances the ligand binding affinity for lectins,
with no change in lectin binding specificity.
saccharide binding sites are more effective multivalent in-
hibitors than those which fail to engage divalent binding. We
further investigated the effects of different linker lengths of s-
and l-20-m-AuNP on Con A binding affinity (Table 1). The two-
times difference in the RIP values of s- and l-20-m-AuNP might
be attributed to the differences in their intrinsic properties, for
example, the orientation of mannose groups on the surface and/
or the rigidity of the linkers.
In conclusion, we have demonstrated that a nanoparticle can
be a good multivalent ligand carrier. The multivalent inter-
actions between m-AuNP and Con A are affected by nano-
particle size and the linker of mannose ligands. Our approaches
may be also applicable to other types of nanoparticles such as
quantum dots24 and magnetic nanoparticles.
We acknowledge financial supports from the NSC
(91-2120-M-003-001), Academia Sinica, NTNU (ORD-
92-03).
Notes and references
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It has been studied that the Con A tetramer presents two
saccharide binding sites on each face, and the distance between
them is 6.5 nm.22 We compared the inhibition potencies of 6-m-
AuNP and 20-m-AuNP in the SPR competition assays (Table
1). As the particle diameters of 6-m-AuNP and 20-m-AuNP are
comparable to or significantly larger than the distance between
two relevant binding sites on Con A, respectively, the mannose
ligands of 6-m-AuNP are less favorable to engage in the
divalent binding of a Con A tetramer than those of 20-m-
AuNP.23 Our system showed that the carbohydrate ligands with
the ability to span the requisite distance to occupy two Con A
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Fig. 2 Inhibition of 0.5 mM Con A binding to the chip by 6-m-AuNP. A set
of inhibition curves for 0, 0.175, 0.5 and 1 mM 6-m-AuNP (top to bottom)
are shown.
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Table 1 The data of dissociation constants (Ki) and relative inhibition
potency (RIP) of carbohydrate-AuNP to Con A (— no inhibition; / not
determined)
Compound
Ki
RIP
aMeMan
20 3 1024
1.0
6-m-AuNP
s-20-m-AuNP
l-20-m-AuNP
6-g-AuNP
6-t-AuNP
8.8 3 1028
2.3 3 1029
3.5 3 1029
1.6 3 1027
—
11.2
127.8
67.5
/
—
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