Biochimica et Biophysica Acta 1760 (2006) 636 – 651

http://www.elsevier.com/locate/bba

Review
Glyconanoparticles: Types, synthesis and applications in glycoscience,
biomedicine and material science
Jesús M. de la Fuente ⁎, Soledad Penadés ⁎
Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC, Isla de la Cartuja, Américo Vespucio 49, 41092 Sevilla, Spain
Received 27 October 2005; received in revised form 30 November 2005; accepted 1 December 2005
Available online 28 December 2005

Abstract

Nanoparticles are the subject of numerous papers and reports and are full of promises for electronic, optical, magnetic and biomedical
applications. Although metallic nanoparticles have been functionalized with peptides, proteins and DNA during the last 20 years, carbohydrates
have not been used with this purpose until 2001. Since the first synthesis of gold nanoparticles functionalized with carbohydrates
(glyconanoparticles) was reported, the number of published articles has considerably increased. This article reviews progress in the development
of nanoparticles functionalized with biological relevant oligosaccharides. The glyconanoparticles constitute a good bio-mimetic model of
carbohydrate presentation at the cell surface, and maybe, excellent tools for Glycobiology, Biomedicine and Material Science investigations.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Glyconanoparticle; Quantum dot; Magnetic nanoparticle; Carbohydrate interaction; Multivalence; Glycobiology

1. Introduction nanoparticles has revolutionized the visualization of cellular

During the last 20 years, there has been a great deal of
interest in the self-assembly fabrication of hybrid materials from
inorganic nanoparticles and biomolecules [1]. As nanoparticles
and biomolecules are of a similar length scale (Fig. 1), it seems
logical that the combination of biomacromolecules to nano-
materials can provide interesting tools for mimicking the
biomolecules which are present at cellular systems, probing the
mechanisms of biological processes, as well as developing
chemical means by handling and manipulating biological
components [2]. At present, it is straightforward to control
and modify the properties of nanostructures to better suit their
integration with biological systems; for example, controlling
their size, modifying their surface layer for enhanced aqueous
solubility, biocompatibility, or biorecognition [2–4]. The use of
gold nanoparticles in biological applications was first high-
lighted in the 1970s with the immunogold staining procedures
[5]. Since then, the labeling of target molecules with gold
⁎ Corresponding authors. Tel.: +34 954489561; fax: +34 95 4460565.
E-mail addresses: lafuente@iiq.csic.es (J.M. de la Fuente),
penades@iiq.csic.es (S. Penadés).
0304-4165/$ - see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2005.12.001
and/or tissue components by electron microscopy. During the
last 10 years, several groups have prepared gold nanoparticles
functionalized with proteins and DNA [1]. These nanoparticles
are being used for assembling new materials, developing
bioassays and as multivalent systems for interaction studies.
Although several reviews on nanomaterials functionalized
with proteins, peptides, DNA and RNA have been published in
the last decade [1,2,6–11], as far as we know, only one of them has
been partially reported on nanoparticles covered with carbohy-
drates (glyconanoparticles) [12]. For this reason, the main aim of
this manuscript is the review of the synthetic methods and
applications of these new hybrid materials made of inorganic
nanoparticles and biologically relevant oligosaccharides.
Carbohydrates are, together with nucleic acids and proteins,
important molecules for life. Much is already known about the
structure, interactions and function of nucleic acids and proteins,
however, the role of carbohydrates in the cell is less clear. The
surface of mammalian cells is covered by a dense coating of
carbohydrates named glycocalyx [13]. In the glycocalyx,
carbohydrates appear mainly conjugated to proteins and lipids
(glycoproteins, glycolipids and proteoglycans) and it is as
glycoconjugates that they develop their biological function.
 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651
Fig. 1. The figure depicts the sizes of nanoparticles in relation to other biological objects.
Now, it is known that these complex oligosaccharides are involved
in the control of many normal and pathological processes [14,15].
A characteristic feature of the biological interactions where
carbohydrates are involved is their extreme low affinity that has to
be compensated by multivalent presentation of the ligands. For this
reason, we developed a new integrated approach based on the use
of nanoparticles that we have named the Glyconanotechnology
strategy [12] to study and evaluate carbohydrate–carbohydrate and
carbohydrate–protein interactions. After our first manuscript on
the application of gold glyconanoparticles to study the self-
aggregation of Lex trisaccharide in the presence of calcium
appeared in 2001 [16], several research groups have developed
different strategies to prepare and apply nanoparticles functiona-
lized with biologically relevant oligosaccharides to study
carbohydrate interactions or to intervene in carbohydrate-mediated
biological processes. After 5 years, there are enough results to state
that carbohydrates are joining proteins, peptides and DNA as
biological partners of inorganic nanomaterials to intervene in
biological processes (Fig. 2).
Fig. 2. Carbohydrates join DNA, proteins and peptides as biological partner of
inorganic nanoparticles.
637
Although numerous examples of gold and magnetic
nanoparticles protected and stabilized with polysaccharides as
chitosan or dextran have appeared in the literature [17–22], this
review is focused on nanoparticles functionalized with
biologically relevant oligosaccharides, where the oligosacchar-
ides confer biological functionality to the inorganic core of the
nanomaterial rather than playing a simple stabilizing role.
2. Types and synthesis of glyconanoparticles
Three different types of nanoparticles functionalized with
oligosaccharides have been reported so far: gold and silver
glyconanoparticles, semiconductor glyco-quantum dots and
magnetic glyconanoparticles.
2.1. Gold and silver glyconanoparticles
Gold nanoparticles are the most stable metal nanoparticles,
and present interesting properties which include a wide array
of assembling model and unexpected size-related electronic,
magnetic and optical properties (quantum size effect). They
already have a number of applications in catalysis and
biology. Therefore, their promises are in these fields as well
as in the bottom-up approach of nanotechnology, and they
will be key materials and building blocks in the 21st century.
While numerous routes exist for the production of colloidal
gold nanoparticles, the method described by Brust et al. [23]
and its variation [24,25] are the most popular synthetic
schemes in the field. As far as we know, most of the
monolayer protected glyconanoparticles have been prepared
by Brust's method. This two-phase synthesis uses a thiol
ligand that strongly binds gold due to the soft character of
both Au and S (which protect the metallic core by a covalent
Au\S bond). A gold salt (AuCl4−) is transferred from a water
solution to toluene using tetraoctylammonium bromide as the
phase-transfer reagent and then reduced by NaBH4 in the
presence of dodecanethiol (Scheme 1) [23]. The organic
phase changes colour from orange to deep brown within a
638 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651
Scheme 1. Synthesis of dodecanethiol gold nanoparticles by Brust's
methodology. (a) HAuCl4, NaBH4, toluene/water, tetraoctylammonium
bromide.
few seconds upon addition of NaBH4. This method allows the
facile synthesis of thermally and air-stable gold nanoparticles
of reduced dispersion and controlled size. Indeed, these
nanoparticles can be repeatedly isolated and re-dissolved in
water without irreversible aggregation or decomposition. The
method can also be carried out in methanol–water solution by
using water soluble thiolates [26] and following this
procedure Murray et al. reported the synthesis of water
soluble gold nanoparticles with the non-natural amino acid
tiopronin as a stabilizer [27].
These nanoparticles protected with alkanethiolates have
received considerable attention due to their stability, suspensi-
bility in different solvents, and facile characterization by
standard analytical techniques. Since Brust reported his
methodology in 1994, several groups have prepared gold
nanoparticles functionalized with peptides, proteins and DNA
[1,2].
Based on this method, we reported the first synthesis of gold
glyconanoparticles by adding a methanolic solution of a
neoglycoconjugate functionalized with a thiol group, to an
aqueous solution of tetrachloroauric acid (HAuCl4) and
subsequent reduction with NaBH4 (Fig. 3) [16]. Following
this method, the synthesis of different glyconanoparticles
functionalized with the tetrasaccharide Le y ([Fucα1–2]
Galβ1–4[Fucα1–2]GlcNAcβ1) [28], the trisaccharide Lex
(Galβ1–4[Fucα1–2]GlcNAcβ1) [16], the disaccharides lactose
(Galβ1–4Glcβ1) and maltose (Glcα1–4Glcβ1) or the mono-
saccharide glucose were reported (Scheme 2) [3]. Different
linkers of hydrophobic (alkanes) and hydrophilic (polyethylene
glycol derivatives) nature were used to bind the carbohydrate to
the gold core [3]. The glyconanoparticles prepared in this way
are water soluble, stable in solution for years, non-cytotoxic and
exceptionally small (less 2 nm). This small size confers them
unusual properties as a permanent magnetism even at room
temperature [29,30].
The same synthetic method has been used by different
groups. Lin et al. [31] have prepared gold nanoparticles
functionalized with glucose, mannose, galactose and manno-
sides; Gervay-Hague et al. [32] with glucose and galactose;
Barchi et al. [33] with Thomsen-Friedenreich disaccharide; and
Kamerling et al. with oligosaccharides involved in the self
recognition process of the marine sponge Microciona prolifera
[34]. The last authors have also prepared a series of gluco- and
manno-oligosaccharide containing glyconanoparticles from free
oligosaccharides by a reductive amination of the saccharides
with trityl-protected cysteamine and further detritylation [35].
Penades' method also provides the possibility to prepare in a
single step, hybrid nanoparticles or nanoparticles with different
density of carbohydrates (Fig. 4). Fluorescence-labeled glyco-
nanoparticles have been prepared by mixing in a determined
ratio a thiol functionalized fluorescein and the neoglycoconju-
gate prior to nanoparticle formation (Scheme 3). Similarly, the
control of the carbohydrate density on the surface of the
nanoparticles, from 5% to 30% in lactose, can be achieved by
mixing different linkers and neoglycoconjugates before nano-
particle formation [3].
Lactose functionalized glyconanoparticles have been also
produced in a three-step procedure by Kataoka et al. [36] First,
gold nanoparticles protected with a poly(ethylene glycol) (PEG)
derivative containing both mercapto and acetal groups was
prepared by Brust's method. The α-acetal-PEG layer formed on
gold nanoparticles was converted to aldehyde by acid treatment
and, in a third step, a reductive amination with p-aminophenyl
glycosides in the presence of (CH3)2NHBH3 was carried out
(Scheme 4). To regulate lactose density on the nanoparticles, this
group has recently [37] used the approach previously reported by
us [3] for the preparation of hybrid glyconanoparticles.
A three-step procedure has also been used by Lakowicz et al.
[38] to prepare hybrid silver glyconanoparticles. They prepared
tiopronin protected nanoparticles which afterwards underwent a
ligand exchange with thiolate boronic acids. The nanoclusters
capped with boronic acids were coupled to polysaccharides
(dextran 3000) and monosaccharides (glucose). However, in
this case, the carbohydrates are not covalently attached to the
nanoparticle.
Another example of non-covalently sugar protected glyco-
nanoparticles was reported in 2004 by Yang et al. [39] for the
synthesis of gold and silver nanoparticles of chitosan and
heparin. This approach uses these polysaccharides as reducing/
Fig. 3. A cartoon showing the mechanism of nanoparticle formation. The
neoglycoconjugates protect the clusters, control their size, and provide
bioactivity.
 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651 639

Scheme 2. Synthesis of Lex, Ley, glucose, lactose and maltose gold glyconanoparticles by Penades' methodology.

stabilizing agents. In both cases the metal nanoparticles were
stabilized by electrostatic interactions between the surface of the
nanoparticle and the polysaccharides. This so-called green
method provides a simple and environmental friendly strategy
to prepare glyconanoparticles without any additional chemical
reducing agent and in aqueous solution.
All the above-mentioned methods provide water-soluble
nanoparticles of reduced dispersion and controlled size. These
nanoparticles are stable, and self-aggregation has not been
observed in any case.
2.2. Glyco-quantum dots
Nanocrystals of semiconducting materials, otherwise includ-
ed in the term quantum dots (QDs), have fascinated physicists,
chemists, and electronic engineers since the 1970s. The most
striking feature of these materials is that their chemical and
physical properties differ markedly from those of the bulk solid
[40]. Since their quantum size effects are understood,
fundamental and applied research on these systems has become
increasingly popular. One of the most interesting applications is
640 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651

the use of nanocrystals as luminescent labels for biological

Fig. 4. The cartoon shows some examples of hybrid glyconanoparticles where
DNA, RNA, lipids, peptides, or biotin coexist with antigenic oligosaccharides.
systems [41–45]. QDs have several advantages over conven-
tional fluorescent dyes: they emit light at a variety of precise
wavelengths depending on their size and have long fluorescent
lifetimes. There are several approaches for the coupling of
biomolecules to semiconductor QDs [46]. Most commonly,
QDs are first prepared at high temperature in the presence of
protective additives to prevent crystal aggregation and to
regulate the growing rate. Alternatively, the protection process
can be performed in solution using thiols with hydrophilic end-
groups to confer stability and water solubility to the QDs. In a
second step, the biomolecules are attached to the protected
nanocrystals for cell labeling. QDs bio-conjugated to peptides
and proteins [42,43,45,47–52], antibodies [43,50–54], DNA

Scheme 3. Preparation of hybrid glyconanoparticles.

 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651
Scheme 4. Preparation of functionalized gold glyconanoparticles by Kataoka's methodology. (a) HAuCl4, NaBH4; (b) H+; (c) NH2-Ph-Lactose, NH2-Ph-Mannose,
(CH3)2NHBH3.
[55–58], and other molecules [59–61] have recently been
prepared mainly by coupling the biomolecules to the thiol
protected QDs and tested as biological markers. However, there
are only few examples of QDs conjugated to carbohydrate
antigens for specific cell targeting.
The first example of QDs protected with polysaccharides was
reported in 2003 by Rosenzweig et al. [62]. This group prepared
CdSe-ZnS quantum dots protected with carboxymethyldextran
and polylysine, and they proved the high affinity of the QDs
toward the glucose binding protein-Concanavalin A (Con A).
Chaikof et al. reported in 2004 the coupling of commercially
available QDs-streptavidin with a biotin end-terminated lactose
glycopolymer [63]. Confocal microscopy confirmed fluorescent
staining of Ricinus communis agglutinin (RCA120)-immobilized
agarose beads due to the glycopolymer–lectin interaction.
We have reported the preparation of water soluble nanoclus-
ters of cadmium sulphide and zinc sulphide covalently bound to
biologically significant neoglycoconjugates by a single step
solution procedure (Scheme 5) [64]. The synthesis of Lex and
Scheme 5. Preparation of glyco-QDs, (a) Cd(NO3)2·4H2O, Na2S, pH 10; (b) Zn
(NO3)2 6H2O, Na2S, pH 10.
641
maltose protected CdS and Zns nanocrystals were carried out.
The procedure was also used for the synthesis of tiopronin and
tiopronin-Tat functionalized QDs which where able to label the
nucleus of human fibroblasts [45].
Fang et al. have recently reported the functionalization with
β-N-acetylglucosamine of CdSe/ZnS core-shell QDs previously
encapsulated with pyridine. The pyridine-encapsulated QDs
were treated with a disulphide derivative of N-acetylglucosa-
mine and NaBH4 in aqueous solution to give water-soluble
glyco-QDs [65].
2.3. Magnetic glyconanoparticles
Magnetic nanoparticles offer exciting new opportunities
including the improvement of the quality of magnetic resonance
imaging (MRI), hyperthermic treatment for malignant cells,
site-specific drug delivery and also the recent research interest
of manipulating cell membranes (Fig. 5) [11,21,22,66,67].
Previously reported iron oxide superparamagnetic nanoparticles
prepared by different surface chemistry have been widely used
for numerous in vivo applications. The biological applications
of these nanomaterials require these nanoparticles to have high
magnetization values, size smaller than 20 nm, narrow particle
Fig. 5. Potential applications of magnetic glyconanoparticles.
642 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651
Fig. 6. Applications of glyconanoparticles.
size distribution and a special surface coating for both avoiding
toxicity and allowing the coupling of biomolecules [21]. Most
commonly, iron oxide nanoparticles are prepared by co-
precipitation of ferrous and ferric salts solution and stabilized
using biocompatible molecules as dextran or oleic acid. In a
second step, the biomolecules are attached by covalent or
electrostatic coupling to the protected nanoparticle. Bio-
conjugated magnetic nanoparticles to peptides, proteins and
antibodies have been developed and tested as biological
markers or in hyperthermia treatments. However, there was
not one example of magnetic glyconanoparticles. Following the
same procedure as for the gold glyconanoparticles, we have
prepared gold–iron nanoparticles functionalized with glucose,
maltose and lactose by adding an iron salt to the aqueous AuCl4−
solution [68]. The so produced nanoparticles are water soluble,
stable and exceptionally small. The properties of these
nanoparticles are particularly interesting as a result of their
quantum size. They show a complex magnetic behaviour with
both paramagnetic and ferromagnetic components. This
anomalous behaviour may be attributed to the gold atoms that
predominate in the metallic cluster with an atomic Au/Fe ratio
from 50:1 to 5:1. Previously, we demonstrated that gold
glyconanoparticles present a permanent magnetism even at
room temperature [29,30]. Work directed to achieve a better
understanding of this phenomenon is currently underway.
The effect of these nanoparticles on human dermal
fibroblasts in vitro has been assessed in terms of cytotoxicity,
light microscopy and scanning electron microscopy [69].
Scheme 6. Structure of GM3, Gg3, lactose and KDNGM3 glycosphingolipids.
3. Application of glyconanoparticles
The glyconanoparticles constitute a good bio-mimetic model
of carbohydrate presentation at the cell surface, opening new
avenues in the field of chemical Glycobiology [12] and
Biomedicine (Fig. 6). In spite of the short history of these
new glyconanomaterials, some applications have already been
reported, focused mainly on the study and evaluation of
carbohydrate interactions. Furthermore, few reports have
appeared on glyconanoparticles as biolabels or biosensors and
some applications in biomedicine or material science have also
been described.
3.1. Glyconanoparticles for the evaluation of carbohydrate
interactions
Three general aspects of glyconanoparticles make them
suitable model systems for the study and evaluation of
interactions where sugars are involved. Firstly, glyconano-
particles are similar in size range to many common biomole-
cules, and for instance they can imitate carbohydrate
presentation in glycoproteins or in glycosphingolipids patches.
Secondly, glyconanoparticles provide a glycocalyx-like surface,
presenting carbohydrates in a globular and polyvalent config-
uration on their surface. Thirdly, glyconanoparticles have
unusual physical properties due to the quantum size effect
[6,29] that can be used for the detection and evaluation of the
interactions.
So far, glyconanoparticles have been used to study both
carbohydrate–carbohydrate (carb–carb) interactions and carbo-
hydrate–protein interactions.
3.1.1. Glyconanoparticles in carbohydrate–carbohydrate
interactions
In 1989, Hakomori proposed that the first phase of cell
adhesion initiates through a carb–carb interaction between
multivalent glycosphingolipid-domains. This initial step is
followed by protein–protein interactions between adhesion
receptors and protein–carbohydrate interactions between
lectins and glycoconjugates. Embryogenesis, metastasis and
other cellular proliferation processes are according to
Hakomori, mediated by carb–carb interactions [70–75].
Several glycosphingolipids involved in carb–carb interactions
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have already been identified. For example, the antigen
determinant Lex seems to mediate morula compaction in
mouse via a carbohydrate self-interaction [70], and the
interaction between the glycosphingolipids GM3 and Gg3
has been proposed to be involved in metastasis of melanoma
cells in mouse [72–74]. It has been demonstrated recently that
the interaction between KDNGM3 and Gg3 is involved in
the binding of sperm to egg membrane in rainbow trout
fertilization (Scheme 6) [75].
Outside from the eukaryotic world, another example where
carb–carb interactions are involved is the species-specific cell
aggregation of marine sponges [76–78]. It seems that a large
extra cellular acidic proteoglycan (g200) is responsible, via a
Ca2+-dependent, homophilic and polyvalent carb–carb interac-
tion, for this aggregation. A sulfated disaccharide and a
pyruvated trisaccharide have been identified as the repetitive
epitopes of the glycan responsible for aggregation [79,80].
Characteristic features of these interactions are their
specificity, their strong dependency on divalent cations, and
their extreme low affinity that has to be compensated by
multivalent presentation of the ligands. The study of carb–carb
interactions faces the important challenge of this extreme low
affinity and attempts to characterize and quantify this
interaction in solution with monomer ligands have usually
failed [81–83]. Multivalent presentation also has been proven to
be important in the study of carbohydrate–protein interactions
[84,85]. However, polyvalence seems even more mandatory in
the case of carbohydrate–carbohydrate interactions.
It was searching for appropriate multivalent systems with
well-defined chemical composition, that we have developed
first gold glyconanoparticles as a tool to imitate multivalent
Fig. 7. Glyconanoparticles for studying carbohydrate–carbohydrate interactions.
643
carbohydrate presentations at the cell surface. Gold glycona-
noparticles functionalized with the trisaccharide Lex (Lex–Au)
have been used to investigate the selective self-recognition of
the Lex antigen via carb–carb interaction (Fig. 7) [12].
Transmission electron microscopy (TEM) has allowed to
observe directly the self-aggregation of the Lex nanoparticles
in 10 mM Ca2+ solution [16]. The TEM micrographs obtained
revealed clear differences between lactose and Lex nanoparti-
cles. The Lex nanoparticles in Ca2+ solution showed large three-
dimensional aggregates at all concentrations, while the lacto
particles remained dispersed as in the water solution. Removal
of the Ca2+ from the solution by addition of EDTA resulted in
the dispersion of the Lex aggregates. Isothermal titration
calorimetry (ITC) has confirmed that this process in solution
is a slow process that takes place with a decrease in enthalpy of
160± 30 kcal mol−1, while the heat evolved in the case of
lactose and maltose glyconanoparticles was very low and
thermal equilibrium was quickly achieved [86].
A similar approach has been followed by Kamerling et al. to
explore the carbohydrate-mediated self-recognition of marine
sponge cells. This group has used water-soluble gold glyco-
nanoparticles coated with a synthetic sulfated disaccharide
fragment as multivalent systems to investigate the g-200
glycan–glycan interaction by TEM [87].
The first kinetics data of Lex–Lex interaction have been
obtained by us using a combination of self-assembled
monolayer (SAMs) of a Lex neoglycoconjugate on a biosensor
gold surface as substrate, Lex gold glyconanoparticles as the
analyte and surface plasmon resonance (SPR) detection [88].
The sensorgrams obtained for the binding of Lex glyconano-
particles to the Lex-SAMs indicated a slow association phase
644 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651
(kon 2.3 ×103 M−1 s−1) and a gradual dissociation phase (koff
1.5 ×10−3 s−1) in the presence of calcium cations. The binding
was of high affinity with dissociation constant in the
micromolar range (Kd, 5.4 ×10−7 M).
3.1.2. Glyconanoparticles in carbohydrate–protein
interactions
Characteristic features of protein–carbohydrate interactions
are high specificity and low affinity. Nature overcomes this low
affinity by clustering of ligands and receptors at cell surfaces
[85], the concept of multivalence becoming an important issue in
glycobiology. For understanding the mechanism of these
interactions, many researchers have developed a variety of
model systems bearing spherical or linear arrays of carbohy-
drates [84,89,90], including glycodendrimers [91–93], glyco-
polymers [94–97], glycopeptides and glycoproteins [98], and
self-assembled systems, e.g., liposomes [74,99], micelles
[100,101] and rotaxane [102]. Some of these multivalent
systems have been shown to be superior ligands for the
binding to endogenous lectins [103,104] and bacterial toxins
such as Shiga-like toxin [105] or cholera toxin [106], drawing
attention to their potential clinical applications. The presenta-
tion and density of the ligand in these recognition processes
have been demonstrated to critically influence the binding
[107–111]. Such an influence is intimately related to the
particular arrangement and architecture of carbohydrate-
binding sites in the receptor.
In the search for new multivalent systems with well-defined
chemical composition, several groups have used gold glycona-
noparticles. The first group using gold glyconanoparticles to
study protein–carbohydrate interactions was Kataoka et al. They
observed the selective aggregation of lactose gold nanoparticles
exposed to RCA120 (Fig. 8) [36] and the ligand density effects on
the aggregation through the evaluations of the interaction
between the lectin and gold glyconanoparticles with regulated
Fig. 8. Schematic representation of the reversible aggregation–dispersion behaviour of lactose gold nanoparticles by sequential addition of RCA120 lectin and
galactose with actual concomitant change in colour from pinkish-red to purple to pinkish-red. Reprinted with permission from ref 36 (Kataoka's group). Copyright
2001 American Chemical Society.
Fig. 9. Schematic illustration of the interactions of glyconanoparticles and ConA
on the biosensor chip in the competition assay. Reprinted with permission from
ref 31 (Lin's group). Copyright 2003 The Royal Society of Chemistry.
density of lactose [37]. This lectin specifically recognizes the β-
D-galactose residue, inducing changes in the absorption
spectrum. The aggregation of the lactose glyconanoparticles
originated a visible colour change from pinkish-red to purple.
This aggregation was reversible and the degree of aggregation
was proportional to lectin concentration. More recently, Chen et
al. have applied the visible colour change for the detection of
protein–protein interactions [112]. Thyroglobulin, a binding
protein for ConA, was added over the mannose-encapsulated
glyconanoparticles and ConA complex. The change from
purple to pinkish-red, due to the dispersion of glyconano-
particles, allowed the qualitatively and quantitatively evalu-
ation of the thyroglobulin–ConA interaction.
The merge between gold glyconanoparticles and SPR
technique have demonstrated to be a powerful tool to evaluate
carbohydrate interactions. Since we reported this combination to
obtain kinetic data for Lex–Lex [88], several groups have used
this approach to quantify carbohydrate–protein interactions. For
example, Lin et al. have used SPR technique to quantitatively
analyze the interaction between mannose gold glyconanoparti-
cles and the Concanavalin A lectin (Con A) [31]. They have
found that the binding has a strong multivalent effect and that the
affinity of mannose glyconanoparticles for Con A could be
adjusted by altering the nanoparticle size or the sugar moiety
 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651 645

(Fig. 9). Recently, Kamerling et al. have evaluated the

interaction between different mannosides and glucosides
prepared from free oligosaccharides and the Con A lectin [35],
demonstrating the multivalent binding character of this
interaction.
Gervay-Hague et al. have reported investigations of
multivalent interactions of galactose and glucose gold
glyconanoparticles with recombinant gp120 [32]. A biotin
NeutrAvidin adhesion assay was used to evaluate the relative
ability of gold glyconanoparticles to displace rgp120 from
plate-bound GalCer. This group found that the gold glycona-
noparticles were more than 300 times more active than the
corresponding dimer.
QDs have been used to evaluate carbohydrate–protein
interactions as well. Fang et al. have recently used the optical
properties of these fluorescent materials to demonstrate the
affinity of β-N-acetylglucosamine-encapsulated QDs with the
wheat germ agglutinin (WGA) by fluorimetry, transmission
electron microscopy, dynamic light scattering microscopy and
flow cytometry [65].

3.2. Glyconanoparticles as biolabels

Gold nanoparticles that are functionalized with proteins

have long been used as tools in biosciences [1,2]. For
example, gold nanoparticles were initially applied in biolog-
ical applications in 1971, when Faulk and Taylor devised the
immunogold staining procedure. The labeling of targeting
molecules with nanoparticles has revolutionized the visuali-
zation of cellular or tissue components by electron microscopy
[5]. In fact, metal, semiconductor, polymers and magnetic
particles functionalized with proteins, peptides, antibodies or
nucleic acids, have been extensively used to target cells
[41,113–119]. However, there are only few examples of
glyconanoparticles used as labels.
Mannose gold nanoparticles have been used to observe the
specific binding to FimH adhesin of bacterial type 1 pili by
TEM [120]. Lin et al. confirmed the specific binding of
mannose nanoparticles to FimH, incubating the nanoparticles
with ORN178 (strain expresses wild-type type I pili) and with
ORN208 (strain is deficient on the fimH gene) and the
binding of mannose nanoparticles to bacterial pili in each
condition was examined by TEM (Fig. 10). The selective
binding of these glyconanoparticles to bacterial type I pili
presented a novel method of labeling specific receptors for
carbohydrates on the cell surface using carbohydrate conju-
gated nanoparticles.
Due to the optical properties of semiconductor nanocrys-
tals, glyco-QDs are potential candidates to be used as cell
labels. The used of QDs for cell labeling has been growing
in the last decade, and it is expected that in the following
years glyco-QDs will be applied in cell biology as a routine
tool to analyze carbohydrate receptors. In fact, Fang et al.
have recently reported the labeling of mice, pigs and sea-
urchin live sperm with CdSe/ZnS core shells QDs
functionalized with β-N-acetylglucosamine or mannose
(Fig. 11) [65]. Acetylglucosamine-encapsulated QDs were
Fig. 10. Typical TEM images of sectioned areas of (A) pili of the E. coli
ORN178 strain bound with m-AuNP, (B) the E. coli ORN208 strain deficient of
the fimH gene without m-AuNP binding. Scale bar = 100 nm. Reprinted with
permission from ref 120 (Lin's group). Copyright 2002 American Chemical
Society.
concentrated at the sperm heads, while mannose-encapsulat-
ed QDs tended to spread over the whole sperm body, due to
the different distribution of the GlcNAc and Man receptors
on the sperm surface.
3.3. Glyconanoparticles for bio-amplification strategies
The emergence of nanotechnology is broadening new
horizons for highly sensitive bioaffinity assays. Metal or
semiconductor nanoparticles functionalized with nucleic acids
and antibodies have been previously used as amplifier for the
detection of DNA and proteins [10]. This use of metal
glyconanoparticles is one of the most promising applications
for this type of materials. As far as we know, there are only
two examples of this application.
Geddes et al. have developed a method to quantify glucose
monitoring the shift and broadening of the plasmon absorption
of gold glyconanoparticles functionalized with a high-molec-
ular-weight dextran [121,122]. The method is based on the
aggregation of the gold nanoparticles with Con A, which results
in a significant shift and broadening of the gold plasmon
absorption. The addition of glucose competitively binds to Con
A, reducing gold nanoparticles aggregation and therefore
decreasing the plasmon absorption when monitored at a near-
red arbitrary wavelength.
646 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651

Fig. 11. Confocal microscope imaging for staining of sperm with glyco-QDs: (A) selective β-N-acetylglucosamine-encapsulated quantum dots labeling on the heads of
sea-urchin sperm (Scale bar = 20 μm), (B) close-up of β-N-acetylglucosamine-encapsulated quantum dots-labeled sea-urchin sperm, and (C) close-up of mannose-
encapsulated quantum dots-labeled mouse sperm. Reprinted with permission from ref 65 (Fang's group). Copyright 2005 Wiley-VCH.

The second example has been showed by Lin et al. [123].
This group has used glucose and Pk (Galα1 →4Galβ1 → 4Glc)
gold glyconanoparticles for the separation and enrichment of
target proteins by incubation with a mixture of proteins (Fig.
12). Using this approach a lectin (PA-IL) was isolated from a
mixture of proteins and detected at femtomole concentrations
using glucose nanoparticles and MALDI-TOF MS analysis.
The methodology allows the identification of unknown target
proteins by in situ digestion with chymotrypsin and the analysis
peptide mass fingerprinting map of chymotryptic peptides.
3.4. Glyconanoparticles in biomedicine
Glyconanoparticles constitute a good biomimetic model to
intervene in carbohydrate-mediated biological processes. For
this reason, glyconanoparticles have been produced and applied
in biomedical applications.
We have recently shown that lactose gold glyconanopar-
ticles maybe potential tools in anti-adhesive therapy [124].
One of the critical steps in metastasis is the adhesion of
tumour cells to the vascular endothelium. After adhesion,
tumour cells transmigrate and create new tumour foci.
Interactions between tumour-associated antigens and epithelial
cell selectins promote tumour cell metastasis. In addition to
this mechanism, carb–carb interactions between glycosphin-
golipids expressed on the tumour and endothelial cell surfaces
also seem to be involved in the critical adhesion step. A
carb–carb interaction between GM3 expressed in a murine
melanoma cell line (B16) and Gg3 or lactosylceramide
(Scheme 6) of endothelium cells has been proposed to be
involved in the first adhesion step of tumour cells to
endothelium before transmigration [72].
Therefore, inhibition of this step by glyconanoparticles that
present carbohydrate antigens expressed either in the tumour
or the endothelium cells might provide effective anti-adhesion
therapy (Fig. 13). Based on the involvement in cell adhesion
of the antigen lactosylceramide, lactose glyconanoparticles
were tested as a potential inhibitor of the binding of
melanoma cells to endothelium. An ex vivo experiment was
designed for the evaluation of the anti-metastasis potential of
the glyconanoparticles. Mice were injected with melanoma
cells pre-incubated with lactose gold glyconanoparticles, and
after 3 weeks, the animals were sacrificed and both lungs
evaluated under the microscope for analysis of tumour foci. A
70% of tumour inhibition was reported as compared with the
group inoculated only with melanoma cells [124].

Fig. 12. The analytical scheme of the Lin's technique for the specific capture of target proteins and the rapid mapping of binding-epitope-containing peptides.
Reprinted with permission from ref 123 (Lin's group). Copyright 2005 Wiley-VCH.
 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651 647

Fig. 13. Possible action mechanism of lactose glyconanoparticles in anti-adhesive therapy. Reprinted with permission from ref 12 (Penades's group). Copyright 2004
Kluwer.

3.5. Glyconanoparticles in material science
The design, synthesis and characterization of surface-
modified nanoparticles are of fundamental importance in
controlling the mesoscopic properties of new materials and in
developing new tools for nanofabrication [125–128]. Self-
assembled monolayers of thiolates on colloidal gold clusters
are therefore ideal substrates for investigating nanoparticle
microfabrication techniques and the effect of surface-bound
reagents on structure–properties relationships [129–131].
We have prepared hybrid gold glyconanoparticles with
fluorescein molecules, with the aim of having a fluorescence
probe for labeling the nanoparticles for biological tests [3].
The inclusion of a fluorescein molecule, attached to the gold
nanoparticle by a thiol chain, gives a hydrophobic character
to the gold glyconanoparticles and also introduces the
possibility of aromatic stacking interactions. The so-prepared
glyconanoparticles formed a superstructure of holes sur-
rounded by nanoparticles as observed by TEM. In the regions
covered by nanoparticles a two-dimensional hexagonal
structure defined by gold dots is observed (Fig. 14) [132].
A centre to centre distance of 6 nm was found, which
corresponds to the length of two consecutive neoglycoconju-
gate molecules. This result suggested that the manipulation of
the molecule attached to the gold glyconanoparticles can
control the organization of the gold clusters opening new

Fig. 14. TEM images of (a) lactose-fluorescein gold nanoparticles and (b) Lex-fluorescein gold nanoparticles, and the proposed model to explain the hexagonal
package. Inset: High-magnification images of the indicated areas.
648 J.M. de la Fuente, S. Penadés / Biochimica et Biophysica Acta 1760 (2006) 636–651
Fig. 15. The glyconanoparticle concept and its potential applications.
possibilities for size-selection devices at the nanoscale by a
“bottom-up” strategy.
In general, metallic nanoparticles are extensively studied
since they exhibit novel electronic, optical, and magnetic
properties. Most of these new properties arise from the so-called
“size effect” which affects the electronic structure, as well as
from the increase of the ratio of atoms located at the surface with
respect to the total number of atoms of the nanoparticle
[133,134]. Glyconanoparticles are not an exception and they
show very interesting physical properties. In fact, we have
reported that 1.4–1.8 nm gold glyconanoparticles functionalized
with thiol derivatives exhibit a localized permanent magnetism
in contrast to the metallic diamagnetism characteristic of bulk
Au or gold nanoparticles functionalized with amminated
derivatives [29,30].
4. Conclusions and perspectives
In this review, we have presented methods to prepare, in an
easy way, a variety of water-soluble and stable glyconanopar-
ticles. All these nanomaterials are good biomimetic models of
carbohydrate presentation at the cell surface.
As mentioned above, the main application of these new
multivalent systems is the study of carbohydrate-mediated
interactions opening new avenues in Glycobiology. Though
the simple and flexible preparation of these glyconanoclusters
and their interesting physical, chemical and biological
properties allow them to extend their utility in Biomedicine,
Biotechnology and Material Science, their use as biolabels or
biosensing has not been extensively developed. We expect that
a variety of applications of glyco-quantum dots, gold
glyconanoparticles and magnetic glyconanoparticles will be
developed in the near future (Fig. 15).
Furthermore, the control of glyconanoparticle composition
using different carbohydrate and non-carbohydrate ligands
would allow to design and to prepare a great variety of hybrid
glyconanoparticles with increasing utility in drug delivery to
generate “multi-action” drugs or as a new vaccine platform,
going from glyconanoparticles to “artificial nanocells”.
Acknowledgements
We thank the colleagues in the Carbohydrate Group who
have decisively contributed to the glyconanoparticles develop-
ment. They are cited in the corresponding references. We thank
also Prof. M. Martin-Lomas for his support and fruitful
discussions. This work was supported by MEC (Grant
BQU2002-03734) and CSIC (I3P contract).
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