Review

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Glyconanoparticles: multifunctional nanomaterials

for biomedical applications
Metal-based glyconanoparticles (GNPs) are biofunctional nanomaterials that combine the unique physical,
chemical and optical properties of the metallic nucleus with the characteristics of the carbohydrate coating.
The latter characteristics comprise a series of advantages that range from ensuring water solubility,
biocompatibility and stability to targeting properties. The selection of suitable carbohydrates for specifically
targeting biomarkers opens up the possibility to employ metallic GNPs in diagnostics and/or therapy.
Within the vast nanoscience field, this review intends to focus on the advances of multifunctional and
multimodal GNPs, which make use of the ‘glycocode’ to specifically address pathogens or pathological-
related biomedical problems. Examples of their potential application in antiadhesion therapy and diagnosis
are highlighted. From the ex vivo diagnostic perspective, it can be predicted that GNPs will soon be used
clinically. However, the in vivo application of metallic GNPs in humans will probably need more time. In
particular, major concerns regarding nanotoxicity need to be exhaustively addressed. However, it is
expected that the sugar shell of GNPs will lower the intrinsic toxicity of metal nanoclusters better than
other non-natural coatings.

KEYWORDS: antiadhesion agents n biodiagnostics n carbohydrates Isabel García1,

n glyconanoparticles n imaging n nanotoxicity
The design and development of high-quality
nanomaterials for medical applications has
boomed in the last decade, as reflected in the
great number of new journals and recent pub-
lications [1] . Nowadays, nanotechnology is
expected to play a key role in the biomedical
field [2] . Diagnostics, therapy, drug delivery and
Marco Marradi1
& Soledad Penadés†1
‘nanomaterials’ have already been on the mar- 1
Laboratory of GlycoNanotechnology,
ket or in preclinical phases before the onset of CIC biomaGUNE/CIBER-BBN, Parque
Tecnológico de San Sebastián, Pº de
this ‘nanoscience era’. Nanoparticles that are Miramón182, 20009 San
based on noble metals, metal oxides and silica, Sebastián, Spain
†
Author for correspondence:
as well as semiconductor nanocrystals (quan- Tel.: +34 943 005 307
tum dots [QDs]), nanotubes and polymeric Fax: +34 943 005 301
nanoparticles, offer new opportunities for bio- spenades@cicbiomagune.es

tissue engineering are the main biomedical areas
in which the application of nanotechnology is
creating a high expectation of health benefits.
The development of new nanotechnology-based
solutions leading to breakthroughs in medicine
requires international synergies, interdisciplin-
ary works and academy–industry collaborations.
Among the numerous worldwide initiatives, the
Nanomedicine European Technology Platform
(ETP Nanomedicine) [101] , the US-NIH [102]
and other European partnerships  [103] have
recently proposed strategic agendas to start a
road-mapping process in the nanomedicine field.
At present, the nanomedicine products on
the market mainly consist of proteins or aptam-
ers functionalized with polyethylene glycol
(PEGylated), nanocrystalline formulations,
micelles and lipo- or viro-somes encapsulat-
ing active small molecules for drug delivery,
nanocomposites for dental or bone repair,
and iron oxide nanoparticles for in vivo imag-
ing [3] . Notably, many of these materials (e.g.,
liposomes and micelles) that are now termed
medical applications. Among them, metallic
nanoparticles display unique magnetic, electri-
cal, optical, mechanical and chemical proper-
ties, which can be tuned by controlling the size
and shape at a molecular level (~1–100 nm) and
by varying the core materials [4] . These unique
physical properties at the nanoscale range are
also being used to engineer nanoparticles for
therapeutic hyperthermia, to release drugs by
light absorption or to obtain images of organs
at very high resolutions. In addition, metallic
nanoparticles might coassemble diverse mol-
ecules on their surfaces with multiple chemi-
cal functions to integrate different tasks in the
same platform, from targeting tissues or cancer
cells for delivering drugs to enhancing image
contrast for diagnosing diseases. The advantage
of metallic nanoparticles towards polymeric or
macromolecular nanoparticles is dimmed by
a major concern regarding the risks associated
with the nanometric size of nanoclusters in terms
of toxicity towards the environment and living
systems [5–8] . Nevertheless, the multifunctional

10.2217/NNM.10.48 © 2010 Future Medicine Ltd Nanomedicine (2010) 5(5), 777–792 ISSN 1743-5889 777
Review García, Marradi & Penadés
integration of many therapeutic and diagnostic
tasks in a single metallic cluster is a challenge to
engineering biofunctional hybrid nanoparticles
for biomedical applications [9] . The integration
of nanotechnology with biomolecules (nano-
biotechnology) is a viable strategy to fulfill the
exigency of biocompatibility and the need for
targeting specific organs, pathogens or over­
expressed proteins in abnormal physiological
processes [10–12] . The resulting hybrid nano­
biomaterials can potentially be applied in d����iag-
nostics, therapy and/or drug delivery, although
up to now nanoparticles of this type have not
been clinically approved.
Peptides, proteins and nucleic acids have
been extensively used to construct biofunctional
hybrid nanomaterials, while research on carbo-
hydrate-based materials lags far behind that of
the other major classes of biomolecules. Most
of the examples of biomedical applications with
glyconanomaterials reported so far make use
of structural polysaccharides as biocompatible
coatings of metallic nanoparticles or as carriers
in drug-delivery systems. This is the case with
dextran- and carboxydextran-coated magnetic
nanoparticles such as Resovist ® (Cliavist™),
Feridex ® (Endorem™) and Sinerem®, which
have been, or still are, in clinical use as contrast
agents, or polysaccharide-based nanomateri-
als such as alginate [13] and chitosan nanopar-
ticles [14] , which are in preclinical development
as carriers for drug delivery in therapy against
cancer. Excellent reviews have already been pub-
lished focusing on the preparation and appli-
cation of polysaccharide nanoparticles as car-
riers  [15,16] . Polysaccharides act as bioadhesive
material in these nanoparticles (increasing drug
loading, prolonging residence time and enhanc-
ing biocompatibility). By contrast, glycans that
serve multiple functions, ranging from assisting
the correct folding of nascent proteins to deter-
mining trafficking of cells in the circulation,
have been scarcely considered in the preparation
of biofunctional nanomaterials. This article will
mainly focus on metallic nanoparticles function-
alized with biologically significant carbohydrates
(glycans), which are essential constituents of
glyco­proteins and glycolipids involved in cellular
recognition processes [17] .
Functional role of carbohydrates
The dense coat of carbohydrates (‘the glyco­
calyx’) on the surface of mammalian cells has
both structural and functional roles in cells and
tissues. In the last 60 years, a significant effort
has been made to isolate and characterize the
778 Nanomedicine (2010) 5(5)
glycan structures of glycosphingolipids (GSLs)
and glycoproteins, and to understand their func-
tional role in key biological processes of cell life,
such as adhesion, growth, signal transduction
and recognition [18] . The complexity in shape,
functionality and dynamic properties of the
constituent glycans of glycoconjugates (glyco-
proteins, glycolipids and proteoglycans) allows
these molecules to function in intermolecular
interactions as encoders (glycocodes) of bio-
logical information. Carbohydrate recognition
is an integral part of normal biological devel-
opment [19] and of the immune defense against
pathogens via the identification of exogenous
carbohydrates [20] . However, many bacterial
and viral pathogens initially adhere to host tis-
sues by binding specifically to carbohydrates
on the host’s cell surfaces. Carbohydrates can
thus be used to target specific diseases or as anti­
adhesion agents and, therefore, the development
of carbohydrate-based drugs is one of the most
stimulating challenges in current research [21,22] .
Currently, carbohydrates find application in
medicine as anticoagulants (heparin and deriva-
tives), antibiotics (aminoglycosides), antidiabe-
tes drugs (especially a-glucosidase inhibitors)
and vaccines. In the field of carbohydrate-based
vaccines, the recent advances in the chemical
synthesis of complex neoglycoconjugates have
given a notable contribution to the develop-
ment of clinical applications. The group of
Vérez-Bencomo in Cuba has recently devel-
oped the first approved human vaccine against
Haemophilus influenzae type b based on a syn-
thetic carbohydrate [23] . The ‘synthetic’ strat-
egy complements the traditional use of bacterial
capsular polysaccharides as vaccines. Other out-
standing examples of carbohydrate-based anti-
bacterial, antiviral, antiparasitic or a­nticancer
vaccines have been extensively compiled [24] .
Glycomaterials
At the heart of chemical glycobiology is the
preparation of glycomaterials that bridge the
efforts to obtain synthetic complex oligo­
saccharides (glycans) with the chemistry of
materials. Several glycomaterials (Figure 1) have
been developed to isolate and purify carbohy-
drate antigens, to study carbohydrate interac-
tions and to apply them in biomedicine (diag-
nosis and therapy). This ranges from the affinity
columns (with the carbohydrate determinants
of human ABO blood groups) to micelles, den-
drimers and liposomes decorated with glyco-
structures. Glycomicroarrays (chips for high-
throughput screening) and glyconanoparticles
future science group
 Glyconanoparticles: multifunctional nanomaterials for biomedical applications Review
Affinity chromatography Drug carriers

O
O

O
O
O
O

O
O
O
O
O

O O
O
O
O
O O
O

O
O
O
O

O
O

O
O
O

O
O
O

O
O
Glycobeads Glycoliposomes Glycodendrimers

Screening Diagnostic and therapy

O
O

O
O
O

O
O

O
O

O O O O

O
O
Metal
O

O
O

O
O

O
O

O
O
Glycoarrays Glyconanoparticles
Metal: Au, Ag, FeAu, CdS...

Figure 1. Examples of different multivalent glycomaterials.

(metallic, magnetic and semiconductor) belong
to the newest generation of micro- and nano-
materials for deciphering the specific roles of
biologically significant carbohydrates [25] . A
comprehensive review by Chabre and Roy has
recently been ­published, describing both the
synthetic strategies to prepare glycodendrimers,
glycofullerenes, glyconanotubes and other gly-
cosylated self-assembled systems, and their main
biomedical applications [26] .
Metallic nanoparticles, biofunctionalized with
carbohydrates (glyconanoparticles [GNPs]),
offer potential applications in the emerging area
of nanomedicine. GNPs and the methodology
developed to prepare them (glyconanotech­
nology) have led to the development of multi­
valent, biocompatible and multifunctional
gold, magnetic and semiconductor GNPs [27] .
They have been used as model systems to study
and intervene in carbohydrate-mediated inter-
actions  [28] , and as magnetic and fluorescence
probes for cellular labeling and targeted imaging.
Most of the studies with these metal-based
glyco­n anotools are in the frame of ‘basic
research’. To the best of our knowledge,
there are no examples of them in clinical use.
Nevertheless, these systems have been shown to
be powerful chemical tools for profiling carbo­
hydrate–carbohydrate, carbohydrate–protein
and carbohydrate–pathogen interactions [29] .
GNPs constitute a good biomimetic model of
carbohydrate presentation at the cell surface,
opening new avenues in the field of chemical
glyco­biology. Moreover, the potential of gly-
conanotechnology also allows the manipulation
of both the organic shell and the metallic core
to obtain nanobioconjugates with magnetic and
semiconductor properties. A plethora of potential
applications in biomedicine and material science
can be f­oreseen using these new biomaterials.
Although, as previously mentioned, there
are significant works on nonmetallic nanopar-
ticles based on polysaccharides [15] , we illustrate
herein mainly examples of metallic GNPs in

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Review García, Marradi & Penadés
which carbohydrates are ‘covalently’ linked to a
nanosized metallic nucleus. These examples were
selected to stress the potential applications and
the importance of biologically relevant carbo­
hydrates in nanomedicine. The methods for
preparation and characterization of ­GNPs are
accounted ­elsewhere [27,29] .
GNPs as tools in
biomedical applications
Why use ‘glyco’ in nanomaterials? There are
several reasons for constructing hybrid nano-
materials with carbohydrate molecules. The
first reason is that the glycocalyx covers the
most external part of eukaryotic cells and con-
trols the initial steps of cell communications by
specific carbo­hydrate-­mediated interactions [30] .
The second reason is that cells need sugars to
survive. Therefore, the uptake of glyconano-
materials by cells should be favored relative to
other non-natural polymers (polyethylene glycol
or synthetic copolymers). A third reason is the
high solubility of simple (monosaccharides and
disaccharides) and complex (oligo- and poly-
saccharides) carbohydrate molecules in water.
Gold nanoparticles capped with simple mono-
saccharides (glucose, galactose or mannose) are
highly soluble in water and stable for years in
solution. Furthermore, endogenous and simple
carbohydrates minimize nonspecific adsorptions
and avoid immunogenic responses [17] .
Thus, the integration of carbohydrates in
nanoparticulated metallic clusters results in a
fruitful symbiosis. Carbohydrates provide bio-
compatibility, bioavailability and biological
functionality to the resulting nanomaterials,
and ensure solubility and stability in biologi-
cal media, while the metallic nanocluster offers
the possibility to coassemble multiple copies of
carbohydrates at a reduced surface, producing
high local concentrations, which can result in
enhanced effects.
In contrast to protein and antibody inter­
actions, which are mainly monovalent and of high
affinity, carbohydrate-mediated interactions are
multivalent and of low affinity. Therefore, when
designing and engineering carbo­hydrate-based
nanotools for biomedical applications, an essen-
tial issue to take into account is the low affinity
and multivalent character of carbohydrate bio-
logical interactions. The multi­valent presenta-
tion of carbohydrate antigens onto the nanopar-
ticle surface in tandem with the possibility of
inserting multifunctionality and multi­modality
by means of n­anotechnology make these novel
systems very attractive.
780 Nanomedicine (2010) 5(5)
The need for a multivalent presentation for
studying self-interactions between carbohy-
drates led to the first preparation of GNPs [31,32] .
Gold GNPs capped with multiple copies of tri­
saccharide determinant, Lewis X, were prepared
to mimic the patches of the Lewis X GSL at the
cell surface and to demonstrate that interactions
between carbohydrate antigens may be a mech-
anism of cell adhesion  [28] . This biologically
inspired approach has provided the basis of the
first generation of metallic GNPs. Gold GNPs
are water-soluble biofunctional nano­clusters with
a 3D polyvalent carbohydrate display and globu-
lar shape, chemically well-defined composition
and an exceptionally small core size [31] . They
can display a large number of carbohydrates on
a reduced surface with a high local concentration
of sugars (100 molecules on approximately 2 nm
core gold) [32] . In addition, GNPs with a lower
density of carbohydrates can also be prepared by
introduction of other selected molecules onto the
gold surface [33] .
Metallic GNPs have the potential to extend
the diversity of biofunctional hybrid materi-
als for biomedical applications. The ability of
carbo­hydrate antigens to function at the cellular
and molecular level together with the physical
properties of a metal core (noble, magnetic or
semiconductor metal) has allowed the appli-
cation of GNPs as antiadhesive agents and
molecular probes in preclinical therapy and
diagnostics (live-cell and in vivo animal imag-
ing). The most significant examples of applica-
tion of GNPs known to date in antiadhesion
therapy and diagnostics will be highlighted in
the following sections.
GNP-based therapy:
antiadhesion agents
Glycan structures in glycoproteins, GSLs and
proteoglycans are the most exposed molecules at
the cell surface. They are involved in adhesion,
growth and signaling [18] , but are also involved in
pathological processes. The early steps of many
pathological processes and pathogen infections
of host cells consist of adhesion processes medi-
ated by multivalent protein–carbohydrate [34]
and carbohydrate–carbohydrate interactions [35] .
The strategy for applying GNPs in antiadhesion
therapy is based on blocking these multivalent
interactions between carbohydrates involved in
adhesion with host-cell or pathogen receptors.
In the case of protein–carbohydrate interactions,
the GNPs mimic the pathogen presentation of
the carbohydrates in order to block host-cell
receptors involved in the recognition process
future science group
 Glyconanoparticles: multifunctional nanomaterials for biomedical applications
and avoid microbial adhesion, or to displace
already-bound microbes from the host cells by
competing with them. In the case of carbohy-
drate–carbo­hydrate interactions, the GNPs are
targeted to directly neutralize the pathogen.
Antimicrobial and anticancer agents based on
GNPs are not commercially available as far
as we know, but some promising in vitro and
ex vivo examples, both with metallic (especially
gold and silver) and nonmetallic (polymeric)
g­lyconanomaterials, have been described.
Multivalent gold GNPs coated with the
disaccharide lactose, designed to mimic the
presentation of the GSL lactosylceramide at the
surface of endothelial cells, were successfully
tested as antitumoral agents in mice [36] . GSLs
work as adhesion molecules, which facilitate
tumor metastasis and/or modulate signaling for
tumor cell growth and motility. It is also known
that changes in the glycocalyx may be associ-
ated with oncogenic transformation as some
GSLs are overexpressed on the surface of tumor
cells and can be targeted as tumor-associated
antigens by specific antibodies [18] . For these
reasons, carbohydrates have been envisioned as
potential tools for antiadhesion strategies and
vaccine development in cancer therapy  [37] .
The interactions between tumor-associated
carbohydrate antigens and epithelial cell pro-
teins (selectins) promote tumor cell metasta-
sis [38] . Carbohydrate–carbohydrate interactions
between GSLs expressed on the tumor and
endothelial cell surfaces also seem to be involved
in the critical adhesion step of tumor cells to the
vascular endothelium [39] . The main GSLs in
lung endothelium are lacto­sylceramide, lacto-
neotetraosylceramide, g­a ngliotriaosylceramide
and globoside [39] .
Based on this information, multivalent gold
GNPs mimicking the presentation of GSL
patches expressed on endothelial cells were pre-
pared to inhibit the adhesion step in a model
system based on a melanoma cell line (B16)
that generates aggressive metastasis in mouse
lungs [36] . GNPs coated with the disaccharide
lactose (lactose-GNPs) were tested as a potential
inhibitor of the binding of melanoma cells to
endothelium cells. GNPs functionalized with
the monosaccharide glucose (glucose-GNPs),
which are not involved in this adhesion pro-
cess, have also been prepared as control systems
(Figure 2) . Preincubation of B16 melanoma cells
with lactose-GNPs and further inoculation to
mice inhibited lung metastasis progression up to
70% in comparison with mice that were injected
only with melanoma cells or with melanoma
future science group
Review
cells pretreated with glucose-GNPs, as demon-
strated by postmortem analyses of tumor foci
in lungs. The results indicate that gold GNPs
might mimic GSL patches at the cell surface and
work as antiadhesion tools that block the spread
of tumor cell metastasis.
An antiadhesion strategy with multivalent
GNPs has also been applied to avoid virus and
bacterial adhesion to host cellular receptors. An
important challenge in modern medicine is the
fight against HIV, which is the etiologic agent
of the worldwide pandemia, AIDS. The genera-
tion of anti-HIV systems (microbicides and/or
vaccines) capable of eradicating the virus is still
an unaccomplished task, in spite of the great
efforts to find a cure for this disease [40] . One
of the striking characteristics of HIV is that
the outer envelope glycoprotein, gp120, is heav-
ily glycosylated by high-mannose glycans. It is
known that this extensive carbohydrate coating
is implicated in virus–host biology, for example
virus transmission [41] and immune response [42]
to infection.
Gold GNPs have been used to investigate the
role of multivalent interactions of HIV envelope
glycoprotein gp120 with host cellular receptors.
Based on works that demonstrated how a path-
way of HIV infection occurs through gp120
interactions with the cellular receptor galac-
tosyl ceramide (GalCer), a GSL expressed on
some mucosal membrane cells, Gervay-Hague
and coworkers [43] have used galactose- and
glucose-containing GNPs as model systems for
studying the multivalent interactions of gp120
with GalCer. Biotinylated GalCer was bound
to NeutrAvidin™-coated polystyrene plates
and recombinant gp120 was then introduced.
In the next step, gp120 was competed off by the
introduction of different carbohydrate-function-
alized nanoparticles and the extent of competi-
tion monitored by absorbance. This adhesion
assay demonstrated the ability of glucose- and
galactose-GNPs to displace gp120 from GalCer
better than the corresponding monovalent sys-
tems, including biotinylated GalCer. These
gold GNPs can thus be considered as interfer-
ing agents in the interaction between GalCer at
host cell surfaces and HIV gp120 with potential
anti-HIV activity.
Another main pathway of HIV infection is by
sexual transmission, which is mediated by anti-
gen-presenting cells at the mucosal endothelium.
The high-mannose glycan clusters of gp120 pro-
mote HIV infection by their interaction with
the C-type lectin dendritic cell (DC)-specific
ICAM  3-grabbing nonintegrin (DC-SIGN)
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Review García, Marradi & Penadés

HO OH OH
OH
OH
O HO OH
OH HO O OH
HO
OH OO O
H OH OH
HO
OH O
O OH
OH H O HO
HO O
HO HO OH O
O O OH
O OH HO
OH O O
HO O OH
HO
O
OH OH O OH HO
OH OH O O
O OH
HO O HO O
O O HO
OH OH
H
OO O
OH OH
O OH
OH OH O O
OH HO OH
HO
HO O
HO
O OH O O
O OH OH
OH O HO
O O
HO
OH S S SS
5 min, 37°C OH OH
O HO
OH
S
S
Au
S
S
S
O O S
HO O
OH
OH

B16F10 cells GNPs Lacto-Au

OH

Control GNP-treated HO
O
O O
B16F10 B16F10 HO O 2 S Au
OH
1000 rpm, r.t.
Gluco-Au

G (gluco-Au)
TC L30 (lacto-Au)
1 week
3 weeks L90 (lacto-Au)
Mouse lungs
Cell viability coinjected with:
1. B16 cells
2. B16 cells + gluco-GNPs
3. Negative control
Lung tumoral foci score/ 4. B16 cells + lacto-GNPs
anatophatologic studies

Figure 2. Gold glyconanoparticles as multivalent tools in antiadhesive therapy. (A) Schematic representation of ex vivo
experiments for evaluating the antimetastatic potential of lacto-GNPs. (B) Schematic representation of lacto-GNPs and gluco-GNPs.
(C) The specific antimetastatic effect of lacto-GNPs. Lungs corresponding to mice treated with B16F10 cells (C1), gluco-GNPs (C2) or
lacto-GNPs (C4) in comparison with the lungs obtained from a control animal (not injected with B16F10 cells; (C3)).
Lacto: Lactose; Gluco: Glucose; GNP: Glyconanoparticle; TC: Tumor control (positive group of mice).
Adapted with permission from [36] .

expressed on DCs [44] . DCs, a main group of
antigen-presenting cells, migrate to the lymph
nodes and once there efficiently transfer the virus
by trans-infection to T lymphocytes, where viral
replication occurs [45] . Mimicking the cluster
presentation of the oligomannosides on the sur-
face of the virus is a strategy for designing car-
bohydrate-based antiviral agents. Penadés’ group
has designed and synthesized gold GNPs coated
with selected oligo­mannoside motifs (manno-
GNPs) mimicking the presentation of the high-
mannose glycans of gp120 [46] . Manno-GNPs
were able to inhibit the DC-SIGN-mediated
HIV trans-infection of human-activated periph-
eral blood mononuclear cells at nanomolar con-
centrations in an experimental setting, which
was designed to mimic the natural route of virus
transmission from DCs to T lymphocytes [47] .
This result proves that synthetic manno-GNPs
could prevent viral attachment to DC-SIGN-
expressing cells and function as an antiadhesive
barrier at an early stage of HIV infection.
Similar antiadhesion strategy can be applied to
avoid attachment of bacteria to the host receptor
cells. Eschericha coli express a mannose-specific
protein (type 1 fimbriae) in the form of elong­
ated, multisubunit appendages, which interact
with glycoprotein and glycolipid receptors on
host cells. In 2002, Lin et al. used mannose-
GNPs to specifically bind to FimH adhesin of
E. coli type 1 pili [48] . They have developed a new
method of labeling specific receptors on the cell
surface by transmission electron microscopy for
the selective differentiation of bacterial strains.
A further step was taken by El-Boubbou et al.
by using a combination of silica-coated mag-
netite nanoparticles with a multivalent display
of mannosides to produce an efficient biosens-
ing method for fast detection and microbial
decontamination without time-consuming
cell culturing [49] . The mannose-coated mag-
netite nanoparticles were able to detect up to
10 4  cells/ml within 5  min and remove up to
88% of E. coli from the medium. The detection

782 Nanomedicine (2010) 5(5) future science group

 Glyconanoparticles: multifunctional nanomaterials for biomedical applications
and identification of pathogenic microorgan-
isms both in the environment and in living
systems by means of nanomaterials is an emerg-
ing applicative field of nanotechnology  [50] .
Carbohydrate-based nanomaterials as antiadhe-
sion multivalent systems may provide a poten-
tial solution to the increasing drug resistance of
pathogens and a valid alternative/complement
to classic antibiotics.
Another strategy to fight pathogens makes
use of silver GNPs. Silver nanoparticles are
in use as antimicrobial systems in interesting
healthcare applications. Silver ions and silver-
based compounds display low toxicity against
human cells, but they are usually lethal against
prokaryotes [50] . For example, Acticoat™ is the
name of a range of antimicrobial barrier dress-
ings for wound care that are based on a nano-
crystalline coating of silver [104] . The synergistic
combination of carbohydrate shell and silver
core nanoparticles has been attempted in order
to develop new microbe-killing agents. Mono-
(glucose and mannose) and di- (lactose and
maltose) saccharide-coated silver nanoparticles
of diameters ranging from 25 to 50 nm dem-
onstrated antimicrobial activity (both growth
inhibition and killing) against different types
of bacteria [51] . Among these GNPs, the 25 nm-
sized particles coated with maltose displayed the
highest antibacterial activity, probably owing
to their smaller size compared with the 50 nm
nanoparticles and their increased presence both
at the cell surface and inside the bacteria. Unlike
these simple sugars, the natural polysaccharide
chitosan and its derivatives display antimicrobial
activity owing to their cationic nature, which
causes cell-membrane disruption and cytoplasm
leakage [50] . Chitosan nanoparticles [52,53] and
chitosan-based silver nanoparticles (6–8 nm in
diameter) have been tested against bacteria [54] .
In the latter case, the intent was to take advan-
tage of the simultaneous double mechanism of
antimicrobial activity due to the bactericidal
effect of nanometric silver and the cationic effect
of chitosan. A direct comparison between the
different results is difficult owing to the differ-
ent assay conditions for the determination of
antibacterial activity, and the diverse size and
functionalization of nanoparticles. However,
it can be concluded that silver GNPs, chitosan
nanoparticles and chitosan-based silver nanopar-
ticles show bactericidal properties. Their mini-
mum inhibition and bactericidal concentrations
are in the µg/ml range, which is an improvement
over simple chitosan and comparable with ionic
silver for certain bacterial strains.
future science group
Review
GNP-based diagnostics
„„
In vivo-targeted imaging
The continuous progress in medical imag-
ing technologies (magnetic resonance imag-
ing [MRI], ultrasound, x-rays and positron
emission tomography) provides novel digital
and powerful systems that are revolutionizing
medicine. Enhancing the spatial resolution and
the contrast of images is critical for improving
the in vivo detection of diseases at early stages.
Biofunctional nanomaterials that can enhance
the contrast and specifically target a disease or
a pathological process are of key importance to
advances in sensitivity and selectivity of diag-
nostics. Some examples of magnetic GNPs
(MGNPs) as MRI contrast agents have been
developed for diagnostic purposes [55–58] . These
carbohydrate-based MRI contrast agents were
designed to specifically address a molecular target
and allow an increase in the local c­oncentration
of contrast agents via precise binding.
One of the most important challenges in
imaging of brain diseases is the detection of
early-stage pathologies, which usually implies
that the blood–brain barrier (BBB) is intact.
In order to address the problem of detection of
tumors in the brain, our group designed and
prepared paramagnetic gold GNPs coated with
different glycoconjugates and suitably chelat-
ing Gd(III) agents [57] . Glucose was chosen
as the sugar able to cross the BBB. Galactose
and lactose were used as sugar candidates to be
preferentially captured by the liver. Preliminary
in  vivo imaging of glioma (generated with
GL261 tumor cells) in mice with the paramag-
netic gadolinium-based gold GNPs indicated
that at the same Gd(III) concentration of glu-
cose-GNPs were able to enhance the contrast
in the tumoral zones better than in clinically
used contrast agents. Lactose-GNPs highly
enhanced the contrast outside the brain, but
they did not seem to reach the tumor, probably
owing to asialoglycoprotein receptor-mediated
liver uptake. The glucose-GNPs were designed
to potentially help reach the CNS by means of
glucose transporters to detect low-grade brain
tumors in which the BBB is not damaged.
Glucose was also the sugar selected in glyco-
peptide Gly-l-Phe-d-Thr-Gly-l-Phe-l-Leu-l-
Ser(O-b-d-glucose)-CONH 2 conjugated to
nonmetallic nanoparticles made of poly(d,l-
lactide-co-glycolide) (PLGA) [59] to increase
penetration in the brain. These works are just a
part of the numerous studies that have tried to
solve the enigma of crossing the BBB for drug
delivery, a field in which the development of
www.futuremedicine.com 783
Review García, Marradi & Penadés
selective and specific treatments and/or diag-
nostic tools able to penetrate the intact BBB
plays a key role. The symbiosis between carbo-
hydrates and nanotechnology (glyconanotech-
nology) can contribute to extend the variety of
bionanomaterials to solve this problem.
Specific targeting of brain tumors has been
achieved by Veiseh et al. with an optical/MRI
nanoprobe [60] . This nanoprobe is comprised of
iron oxide nanoparticles coated with a PEGylated
chitosan, to which the targeting ligand chloro-
toxin and a near-infrared fluorophore Cy.5.5
were conjugated. The magneto-f luorescent
nanoprobe was able to traverse the BBB, spe-
cifically target brain tumors and leave the BBB
uncompromised, as demonstrated by in  vivo
MRI and biophotonic fluorescence imaging.
Iron oxide nanoparticles functionalized with
carbohydrate has allowed presymptomatic
in  vivo imaging of brain disease. The initial
recruitment of leukocytes in brain inflammation
processes takes place across intact, but activated,
brain endothelium. To address the upregulation
of certain types of carbohydrate-binding trans-
membrane proteins (CD62 selectins) in acute
inflammation, van Kasteren et  al. designed
MRI-active GNPs coated with sialyl Lewis X
(sLe x), which specifically bind these protein
b­iomarkers [56] .
In the study, they chose a rat model of brain
inflammation in which the BBB was intact for
in vivo experiments. Dextran cross-linked iron
oxide (CLIO) nanoparticles were used to con-
jugate multiple copies of sLex. Unfunctionalized
CLIO and CLIO conjugated with N-acetyl
lactosamine were used as controls. The mag-
netic sLex-GNP allowed the direct detection of
activated endothelial cells in acute inflamma-
tion by means of MRI (Figure 3) . The success of
the approach of Davis and collaborators was to
visualize the symptoms of a brain disease (inside
the BBB) by labeling the selectin expression on
activated endothelial cells outside the BBB.
Furthermore, the high iron content of the GNPs
improved detection on MRI scans.
Gold GNPs have also been tested in molecular
and cellular optical imaging. Park’s group has
prepared multifunctional gold GNPs capped
with hyaluronic acid labeled with the near-infra-
red fluorescence dye Hylite 647 (HHAuNPs)
(Figure 4) [61] . The paper presents in vivo imaging
of arthritic inflammation and human ovarian
carcinoma (OVCAR-3) tumor in mice upon
systemic injection with the GNPs. The fluo-
rescence quenching by energy transfer between
the dye and the gold surface is deactivated when
784 Nanomedicine (2010) 5(5)
the GNPs reach the target zones (arthritic joints
and tumors), where the synergy between reac-
tive oxygen species and overexpressed hyaluroni-
dase degrade hyaluronic acid moieties. The dye
release allows the ultrasensitive detection of
these disease states by in vivo fluorescence imag-
ing. These results suggest that gold nanoprobes
can be exploited not only as in vitro molecular
and cellular imaging sensors, but also as in vivo
optical imaging agents for detection of local
hyaluronic acid degrading diseases.
In vitro and ex vivo liver optical imaging has
also been achieved by means of semiconduc-
tor nanocrystals capped with sugars (glyco-
QDs) [62] . Galactose-, galactosamine- and man-
nose-QDs were prepared and tested in mice.
Galactose-QDs are preferentially taken up via
asialoglycoprotein receptor-mediated endo-
cytosis in  vitro. Moreover, QDs capped with
mannose and galactosamine were sequestered
s­pecifically in the liver.
In vitro biodiagnostic devices
In vitro assays based on GNPs for detecting dis-
eases or infections (medical diagnostic) are still
in the preliminary stages. Most of the work in
this field is ‘proof-of-principle’ studies based on
model proteins and on the surface plasmon of
the gold as a physicochemical detection method.
Although this research is more accessible and less
risky than in vivo diagnosis, only a few examples
to detect biomarker proteins by using GNPs
(carbohydrate–protein interactions) have been
developed [63–65] . Ban et al. recently described
a simple assay to probe disease-­a ssociated
activity using glycosaminoglycan-assisted syn-
thesized gold nanoparticles [63] . The method
can be extended to test the activity of several
polysaccharide-degrading enzymes, involved in
many pathophysiological alterations in human
diseases, such as tumor progression or neuro-
logical disorders. The detection is based on
the red shift in the plasmon resonance peak of
glycosaminoglycan-protected GNPs caused by
the aggregation when the enzyme cleaves the
polysaccharide chains.
There is no test for accurate diagnosis of
Alzheimer’s disease, the most common form of
dementia among elderly people. Alzheimer’s dis-
ease is correlated with the deposition of amyloid
peptides in the brain of patients. The amyloid
is thus a major target in the search for novel
diagnostic and therapeutic approaches. Sialic
acid-modified gold nanoparticles, supported
on a carbon electrode, have been proposed by
Chikae et al. to detect amyloid-b peptides [64] .
future science group
 Glyconanoparticles: multifunctional nanomaterials for biomedical applications Review
1 2
H2N
H2N N H2
H2N
N H2
H2N
H2N
N H2
N H2
3 4

OH NH
O
HO S
N 5 6
HO NHAc H ×106
GNP-GIcNAc

OH OH NH
HO O O
S
O HO N
OH H ×106
OH
GNP-LacNAc

HO OH
C O2H OH
OH OH NH
O O O
AcHN O S
O HO N
HO OH NHAc H
OH ×106
GNP-siaLacNAc

HO OH
OH C O2H
OH OH NH
AcHN O O O
O S
HO O O N
OH NHAc H ×106
HO
O
OH
OH GNP-sLex
HO

Figure 3. Magnetic glyconanoparticles bearing multiple copies of a glycan ligand of

selectins and their application in presymptomatic in vivo imaging of brain disease.
(A) Synthesis of iron oxide GNPs-sLex from amine-functionalized magnetic nanoparticles and all the
intermediated GNPs (GNP-GlcNAc, GNP-LacNAc and GNP-siaLacNAc). (B) Selected images taken
from the T2*-weighted 3D datasets (1, 3 & 5) and 3D reconstructions of the accumulation of contrast
agent (2, 4 & 6); sLex-GNP enables clear detection of lesions in clinically relevant models of multiple
sclerosis (3 & 4) and stroke (5 & 6) in contrast to unfunctionalized control nanoparticle (1 & 2).
GlcNAc: N-acetyl glucosamine; GNP: Glyconanoparticle; LacNAc: N-acetyl lactosamine; sia: N-acetyl
neuraminic acid; sLex: Sialyl Lewis X.
Adapted with permission from [56] .

The attachment of amyloid-b peptides to the detection using lectin-functionalized Cu/Ni/

sialic acid layer was confirmed by electrochem-
istry and atomic force imaging. The oxidation
signal of a tyrosine residue from the captured
peptide was monitored using differential pulse
voltammetry.
La Belle et al. described a new electrochemical
method for the detection of both natural gly-
coproteins and GNPs thanks to carbohydrate–
lectin-specific interactions [65] . Impedimetric
Au electrode chips was used. GNPs conjugated
to Thomsen–Friedenreich disaccharide, as well
as glycoproteins, asialofetuin and fetuin, were
rapidly detected up to fM range.
Very recently, Huang’s group has developed
iron-oxide MGNPs (the carbohydrates being man-
nose, galactose, fucose, sialic acid and N-acetyl
glucosamine) as nanosensor systems not only to
detect and differentiate cancer cells, but also to

future science group www.futuremedicine.com 785

Review García, Marradi & Penadés

hv hv+
NSET
s s
s s s s
s s s
Oligo-HA ROS/HAdase
s s s s
s s
Liver Heart Lung
O
HilyteFluor™647 HO
O
O H H

H O NH
H H
HO O HO H
HO O
O H H O
H
OH H
O CH3 Spleen Tumor Kidney
HO H
H H
O NH n
O HO
H Color bar
H H Min = 3e + 07
0.5 1.0 1.5 2.0
O CH3 Max = 2e + 09
HO H OH H × 109
H

30 min 3h 6h 24 h 30 min 6h 12 h 24 h

Arthritis Tumor
Color bar Color bar
Min = 1e + 08 1 2 3 4 5 Min = 1e + 08 1 2 3 4 5
Max = 5e + 09 × 109 Max = 5e + 09 × 109

Figure 4. Gold nanoparticles coated with hyaluronic acid as nanoparticle surface-energy transfer-based nanoprobes for
in vivo imaging of rheumatoid arthritis and tumor. (A) Schematic illustration of Hilyte-647 dye-labeled oligo-hyaluronic gold
nanoparticles (HHAuNPs). (B) Fluorescence images of six different organs obtained from tumor-bearing nude mice after the systemic
injection of HHAuNPs. (C) In vivo fluorescence images of collagen-induced rheumatoid arthritis mice upon tail vein local injection of
HHAuNPs. (D) In vivo fluorescence images of tumor-bearing nude mice upon tail-vein injection of HHAuNPs.
HA: Hyaluronic acid; hv: Planck equation; NSET: Nanoparticle surface-energy transfer; ROS: Reactive oxygen species.
Adapted with permission from [61] .

quantitatively profile their carbohydrate-binding MCF-7/Adr-res cells, which caused significantly

abilities by MRI [58] . Incubation of MGNPs with
nine different types of cancer cells resulted in the
specific formation of aggregates, leading to shorter
T2 relaxation time and, consequently, a darkened
MRI image. Unfunctionalized iron oxide mag-
netic nanoparticles were unable to generate sig-
nificant T2 changes and were used as controls.
Despite the simple structures of the monosaccha-
rides utilized, the T2 changes were characteristic
molecular signatures for each cell line and linear
discriminant ana­lysis was employed to differen-
tiate the cell lines. It was also demonstrated that
galactose-MGNPs were able to detect cancer cells
in the presence of normal cells using the MRI
response as a molecular signature. This proof
of principle was realized by using breast cancer
larger T2 changes after binding with galactose-
MGNPs in comparison with the normal breast
endothelial cells 184B5. The importance of this
work resides in that the sugar-binding prefer-
ence for a specific cancer cell line, dictated by
the ‘glycocode’, can be potentially used for cancer
diagnostics. Furthermore, the magnetic nature
of these MGNPs can facilitate cellular enrich-
ment through magnet-mediated separation and,
in perspective, the identification of the involved
carbohydrate receptors.
The development of carbohydrate-based diag-
nostic devices implies a great complexity owing
to the molecular diversity of carbohydrates
and natural polysaccharides. This complexity
confers them a large array of functions of great

786 Nanomedicine (2010) 5(5) future science group

 Glyconanoparticles: multifunctional nanomaterials for biomedical applications
significance that makes difficult the identifica-
tion of new and suitable carbohydrate-interact-
ing biomarkers for diseases, but, at the same
time, enriches the diversity of biofunctional
nanomaterials. Much work with GNPs must still
be carried out in terms of sensitivity, selectivity
and versatility in order to reach the success of
the bio-barcode assays for prostate-specific anti-
gen (DNA and antibody barcodes) developed by
Mirkin and coworkers [66] . However, multifunc-
tional GNPs able to specifically detect carbo-
hydrate-binding receptors and their integration
into novel devices can allow the improvement of
clinical biosensing for diagnosis.
Toxicity & biodistribution of GNPs
As is usually the case when a new technology is
initiated, the future implications of the nano-
technological revolution for society and also the
potential risks to human health and the environ-
ment have to be carefully taken into consider-
ation. As the toxicology of the new nanomateri-
als is mostly unknown, there is a strong need to
corroborate whether these materials have toxic
effects not only to cells but especially to the
human body. This section reports on aspects of
the toxicology of GNPs with the main aim to
raise awareness about the importance, even in
basic research, to verify their possible adverse
effects to living systems. Cell-based in vitro stud-
ies should be considered as a first step in this
direction [67] . In vivo assays, including pharma-
cokinetics and pharmacodynamics, are essential
to assess any toxicological risk of nanomaterials
for future biomedical applications [68] .
It is expected that nanoparticles coated with
carbohydrates that are compatible with, and nec-
essary for, cell growth will present less toxicity
and better biodistribution than nanoparticles
covered with materials that are exogenous to
cells. Examples of cytotoxicity studies of GNPs
have generally demonstrated that metallic GNPs
are not toxic to cells up to concentrations in the
micromolar range. Usually, these studies have
been carried out as preliminary and necessary
tests for further application of GNPs to bio-
logical models. Cytotoxicity of approximately
2 nm diameter gold glucose-, maltose- and lac-
tose-GNPs were evaluated in murine melanoma
B16F10, COS-1, embryonic carcinoma F9 and
NIH-3T3 fibroblast cell lines [36] . At 10 µM,
only maltose-GNPs induced negative effects
when the treatment was prolonged for several
hours, although no toxic effects were observed
at short incubation times (<120  min). Cell
viability of hTERT-BJ1 human fibroblasts with
future science group
Review
the analogous magnetic iron-doped (~0.1–2%)
gold GNPs confirmed that, unlike glucose- and
lactose-MGNPs, maltose-MGNPs demonstrated
a very high toxicity with only 25% of the origi-
nal cells surviving at concentrations higher
than 15 µM  [69] . The effect of maltose is not
unexpected as it is not an endogenous sugar in
cells. Furthermore, in this work, evidence was
obtained demonstrating that the nature of the
carbohydrate molecule on the GNPs can induce
different cellular behaviors. A morphology study
using scanning electron microscopy indicated
different cellular responses to the carbohydrate
on the nanoparticles. No aberrations or morpho-
logic changes were observed at the cell surface
with glucose-MGNPs. Conversely, both lactose-
and maltose-MGNPs were endocytosed by the
cells. In the case of cells treated with maltose-
MGNPs, a high activity was observed on the
cell surface, with formation of lamellipodia
and filopodia.
It was also found that paramagnetic Gd(III)-
based gold GNPs coated with glucose, galactose
or lactose were not cytotoxic to glioma C6 cells
at concentrations as high as 20 µM [57] . Gold
nanoparticles coated with different types of
high-mannose oligosaccharides were not toxic
to Raji DC-SIGN+, to DCs or to human acti-
vated peripheral blood mononuclear cell at
c­oncentrations of 100 µg/ml [47] .
Bioconjugate semiconductor nanocrystals
(QDs) have already been prepared as probes
for labeling cells and for in vivo optical imag-
ing. The size and the bioorganic shell play an
important role in the fate of QDs at the cel-
lular level [70] . Passivation with inorganic shells
(silica-coating or ZnS) or biocompatible organic
shells is required to minimize the intrinsic tox-
icity of these semiconductive nanocrystals [71] .
Carbohydrates have been shown to enhance
the biocompatibility of QDs [62,72,73] . Ongoing
works in our laboratory with glyco-QDs indi-
cate that carbohydrates decrease the intrinsic
cytotoxicity of the inorganic core by one order
of magnitude with respect to the correspond-
ing ‘naked’ QDs. Neoglycoconjugates able to
self-assemble in highly packed monolayers on
the semiconductor surface probably better pro-
tect the metallic core and avoid the release of
toxic metals. In agreement with this observation,
Narain and coworkers noticed that glycopolymer
coatings are able to mask the QDs’ surface in a
more efficient way than small carbohydrates [73] .
In this case, the lower level of toxicity may also
depend on a lower uptake due to the bigger
dimension of glycopolymer-QDs with respect
www.futuremedicine.com 787
Review García, Marradi & Penadés
to the small glyco-QDs. Kikkeri et al. also dem-
onstrated, by measuring serum transaminase
levels in a model of liver injury, that the order
of hepatotoxicity of monosaccharide-capped
QDs is lipopolysaccharide < galactose-QDs <<
galactosamine-QDs [62] .
The in vivo biodistribution and clearance from
the body of nanomaterials have to be carefully
studied before these can be applied clinically. In
general, cell-based assays are not considered to
be clear proof of lack of toxicity. It is not surpris-
ing that recently, the editor-in-chief of a journal
focused on the study of the molecular mecha-
nisms by which toxic agents interact with living
systems proposed a set of guidelines for articles
dealing with nanoparticles for b­iotechnology
applications [74] .
Biodistribution of iron oxide-based nano­
particles capped with dextran or synthetic
polymers has been extensively studied because
these magnetic nanoparticles are already com-
mercialized and in clinical use. The biodistribu-
tion and toxicity profile of chitosan-coated iron
oxide nanoprobes used for specific targeting of
brain tumors [60] was determined by ex vivo near-
infrared signal quantification of excited tissues
(tumor, brain, heart, liver, spleen, kidney and
muscle) of injected mice. Nanoprobe accumu-
lation was observed in liver, but aspartate and
alanine aminotransferase levels did not show
marked elevation compared with noninjected
control mice.
Few works on the effects of metallic GNP
injection to animals and postmortem biodistri-
bution studies have been reported in the litera-
ture. One of them is the qualitative evaluation
of the consequences after intravenous injection
of gold lactose- and glucose-GNPs to mice [36] .
Some of the animals presented bristled hair in
the first days, but no other special behavioral or
pathological effects were noticed. Histological
analyses of several organs did not reveal any sig-
nificant alteration, suggesting that intravenous
inoculation of mice with lactose- or glucose-
GNPs (up to 90  µM) does not promote any
relevant toxic effect in live animals.
A work devoted to biodistribution studies
of metallic GNPs has recently been reported
[75] . In this work, Gum Arabic- and maltose-
stabilized gold GNPs were administered intra-
venously to juvenile swine. Blood, tissue and
urine samples were collected for gold ana­lysis (by
atomic absorption spectroscopy and microscopic
examination) in order to prove that the sugars
were able to direct the nanoparticles to specific
tissues within the body. More than 50% of the
788 Nanomedicine (2010) 5(5)
total gold dose was found in the liver for Gum
Arabic-GNPs and in lungs for maltose-GNPs,
demonstrating that the sugar coating facilitates
targeting of different organs.��������������
�������������
No abnormali-
ties were noted in the biochemical ana­lysis or
m­icroscopic examination of the tissues.
In our preliminary results concerning
in vivo detection of glioma in mice [57] , we have
qualitatively noticed that GNPs were mainly
excreted via the renal system, although evi-
dence of gold accumulation in liver was also
observed [Alcántara D, PhD Thesis, University of
Seville, 2008, Unpublished Data] . The biodistribu-
tion of the aforementioned nanoparticles made
of poly(lactide-co-glycolide) in different organs
(liver, spleen, heart, lungs, kidneys and brain) at
different times is one example of the study of the
fate of polymeric GNPs [59] .
Regarding the application of engineered
nanomaterials in everyday life, and mostly in
nanomedicine, the classic ways of evaluating
toxicity of bulk materials may not be sufficient
to establish all the possible risks associated with
nanoparticles in terms of human health and
the environment. The different chemical and
physical properties owing to the size and surface
characteristics of these nanomaterials and the
incoming bloom of multifunctional and multi-
modal nanoparticles, which will ideally mimic
and work as ‘nanocells’, demand particular care
to ensure their safe manufacturing and use [8] .
The striking features of nanomaterials may thus
hide an Achilles’ heel in terms of safety by poten-
tially generating an unprecedented form of toxic-
ity to biological systems. As a matter of fact, a
new toxicological science (Nanotoxicology – a
journal published by Informa Healthcare – was
launched in 2007) is emerging and a great level of
attention is starting to spread at a basic research
level. It is essential to explore the linkage between
nanoparticle structure and toxicity. It is evident
that there is a strong need for solid guidelines for
testing methods in order to have additional infor-
mation on these substances in the future. The
continuous efforts in terms of legislation related
to the health, safety and environmental aspects
of nanomaterials, regulatory research needs and
related measures and in terms of guidance manu-
als promoted by intergovernmental organisations
emphasize the importance of this question [105] .
Conclusion
Glyconanoparticles modified with biologi-
cally relevant carbohydrates are promising new
nanotools for addressing biomedical problems.
Carbohydrates are present at the cell surface
future science group
 Glyconanoparticles: multifunctional nanomaterials for biomedical applications
and are involved in the first cellular contacts.
For this reason, the implication of carbohy-
drates in nanoparticulate systems has evolved
from a protective to a functional role. The ten-
dency is to explore more deeply the potential
of carbohydrates as molecules encoding bio-
logical information. The higher complexity
and diversity of natural glycans with respect
to other important biomolecules (e.g., DNA
and proteins) make the discovery of specific
carbohydrate epitopes implicated in the recog-
nition processes difficult. However, once these
epitopes are determined, great selectivity and
specificity can be achieved by using them. An
important aspect to be taken into consideration
is that natural carbohydrate epitopes are usually
presented in a multivalent display to compen-
sate the low-affinity monovalent carbohydrate-
mediated interaction. GNPs can mimic the car-
bohydrate clustering at cell surfaces and ensure
a multivalent presentation of the epitopes.
Furthermore, glyconanotechnology allows in
a controlled way the simultaneous incorpora-
tion of different molecules in the same metal-
lic nanocluster to achieve multifunctionality.
Liposomes and polymeric nanoparticles are the
only synthetic drug carriers in clinical use, but
their physicochemical properties restrict their
Review
applications (e.g., low cellular uptake or slow
diffusion into solid tumors). Metallic GNPs
can offer a valid alternative to these systems
by selective targeting and incorporation of
m­u ltifunctionality and multimodality.
Structural polysaccharide-based iron oxide
GNPs are already in clinical use as MRI con-
trast agents. To date, there are no examples of
medical applications of metallic GNPs coated
with carbohydrates. The examples of GNPs as
antiadhesion agents or for in vivo biodiagnostic
imaging described here are based on specific car-
bohydrate-mediated interactions at a molecular
level. These targeted GNPs have an enormous
potential for medical applications.
Future perspective
It is difficult to predict the use of metallic
GNPs in the clinic. The pharmacy industry is
not interested in developing drugs that contain
potentially toxic or nonmetabolizable compo-
nents, such as gold, copper, cadmium or silica,
unless safety data are available. However, when
considered in chemo- or radio-therapeutic treat-
ments, a high risk is acceptable owing to the crit-
ical situation of patients. GNPs that specifically
target tumors and contain curative elements
(antitumoral and antihyperthermia) is a valid

Executive summary
Metal glyconanoparticles: a promising class of biomaterials
ƒƒ Glyconanotechnology allows the straightforward coassembling of multiple functions onto nanoclusters to perform different tasks all
at once.
ƒƒ Integration of carbohydrates in metallic nanoclusters (glyconanoparticles [GNPs]) extends the diversity of biofunctional hybrid materials
for biomedical applications.
ƒƒ Carbohydrates not only enhance water solubility, stability and biocompatibility of the metallic nanoclusters, but confer broad
functionality by means of their complex ‘glycocode’.
ƒƒ The nanosized metallic core provides unique physicochemical properties and offers the possibility to coassemble multiple
copies of carbohydrates at a reduced surface, resulting in high local concentration and multivalency, which are essential in
carbohydrate‑mediated interactions.
Urgent questions about metal GNPs
ƒƒ There is a need for the identification of new and suitable carbohydrate biomarkers for diseases.
ƒƒ Efforts are being made to produce new carbohydrate-based nanoparticles with increased selectivity, sensitivity and versatility for
biomedical applications.
ƒƒ Better biophysical techniques need to be developed for the characterization of the spatial distribution of carbohydrate clustering on
metal surface.
ƒƒ Protocols are needed for the scale-up of GNPs production.
ƒƒ Studies of cytotoxicity, biodistribution and nanotoxicology to assess the real risks to human health and environment are needed prior
to in vivo applications of nanomaterials. It is expected that the sugar shell of GNPs will lower the intrinsic toxicity of metal nanoclusters
better than other non-natural coatings.
Metal GNPs in nanomedicine
ƒƒ Biologically relevant carbohydrates have to be extensively considered in nanomedicine.
ƒƒ Metal GNPs as antiadhesion agents and diagnostic systems have to be translated from the bench to the bed.
ƒƒ Selective and multimodal magnetic GNPs as contrast agents in molecular imaging have to be developed for diagnostic purposes.
ƒƒ Metal GNPs for combining medical imaging techniques and therapy are an important challenge in nanomedicine.
ƒƒ Integration of multifunctional GNPs into novel devices to specifically detect carbohydrate-binding receptors can improve clinical
biosensing for diagnosis.

future science group www.futuremedicine.com 789

Review García, Marradi & Penadés

alternative to chemo- and radio-treatments. antigens and immunogenic peptides have

Regarding the integration of GNPs in biosensors,
the great number of carbohydrate-related targets
enhances the possibility of their development for
ex vivo diagnostics in the clinic. For example, it
is known that no single biomarker can be used
for cancer diagnosis. Multifunctional GNPs car-
rying several carbohydrates and able to detect
more specific carbohydrate-binding receptors
and their integration into novel devices would
improve c­linical biosensing for diagnosis.
Glyconanoparticles could also provide an ade-
quate platform to generate carbohydrate-based
vaccines. The overexpression of tumor-associated
oligosaccharides is exploited to develop antican-
cer vaccines. Polysaccharide–protein conjugates
are being used in vaccination strategies against
bacterial and viral infection. GNPs that coas-
sembled tumor-associated carbohydrate epitopes
and T-cell helper peptides in a controlled way
might induce an immune response. The first
GNPs protected with self-assembled monolay-
ers of different tumor-associated carbohydrate
already been prepared in our laboratory as a
new platform for potential anticancer vaccines;
we are currently working on the immunologic
response of these constructs.
Acknowledgement
The authors thank the members of the Laboratory of
GlycoNanotechnology that along the years have contributed
to the development of glyconanoparticles. Their names
appear in the related publications.
Financial & competing interests disclosure
The present work was supported by the Spanish Ministry of
Science and Innovation, the European Commission, the
Department of Industry of the Basque Government, and
the CIBER-BBN. ��������������������������������������
The authors have no other relevant affili-
����
ations or financial involvement with any organization or
entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript
apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.

Bibliography
Papers of special note have been highlighted as:
n of interest
1 Nanotechnology (Volume 5 – Nanomedicine).
Vogel V (Ed.). Wiley-VCH Verlag GmbH
& Co. KGaA, Weinheim, Germany
(2009).
n One among a multitude of articles and
specialized book series.
2 Riehemann K, Schneider SW, Luger TA,
Godin B, Ferrari M, Fuchs H: Nanomedicine
– challenge and perspectives. Angew. Chem.
Int. Ed. 48, 872–897 (2009).
3 Wagner V, Dullaart A, Bock A-K,
Zweck A: The emerging nanomedicine
landscape. Nat. Biotechnol. 24, 1211–1217
(2006).
4 Hodes G: When small is different: some
recent advances in concepts and applications
of nanoscale phenomena. Adv. Mater. 19,
639–655 (2007).
5 Oberdörster G: Safety assessment for
nanotechnology and nanomedicine: concepts
of nanotoxicology. J. Intern. Med. 267,
89–105 (2010).
6 Suh WH, Suslick KS, Stucky GD, Suh Y-H:
Nanotechnology, nanotoxicology, and
8 Nel A, Xia T, Madler L, Li N: Toxic potential
of materials at the nanolevel. Science 311,
622–627 (2006).
9 Sanvicens N, Pilar Marco M: Multifunctional
nanoparticles – properties and prospects for
their use in human medicine. Trends
Biotechnol. 26, 425–433 (2008).
10 Nanobiotechnology: Concepts, Applications and
Perspectives (Volumes). Niemeyer CM,
Mirkin CA (Eds). Wiley-VCH Verlag GmbH
& Co. KGaA, Weinheim, Germany (2004).
11 Niemeyer CM: Nanoparticles, proteins, and
nucleic acids: biotechnology meets materials
science. Angew. Chem. Int. Ed. Engl. 40,
4128–4158 (2001).
12 Katz E, Willner I: Integrated nanoparticle-
biomolecule hybrid systems: synthesis,
properties, and applications. Angew. Chem.
Int. Ed. Engl. 43, 6042–6108 (2004).
13 Chavanpatil MD, Khdair A, Panyam J:
Surfactant–polymer nanoparticles: a novel
platform for sustained and enhanced cellular
delivery of water-soluble molecules. Pharm.
Res. 24, 803–810 (2007).
14 Park JH, Kwon S, Lee M et al.: Self-assembled
nanoparticles based on glycol chitosan
bearing hydrophobic moieties as carriers for
doxorubicin: in vivo biodistribution and
16 Chiellini F, Piras AM, Errico C, Chiellini E:
Micro/nanostructured polymeric systems for
biomedical and pharmaceutical applications.
Nanomedicine 3, 367–393 (2008).
17 Essentials of Glycobiology (2nd Edition).
Varki A, Cummings R, Esko JD et al. (Eds).
Cold Spring Harbor Lab Press, Cold Spring
Harbor, NY, USA, 1–732 (2009).
18 Hakomori S-I: Structure and function of
glycosphingolipids and sphingolipids:
recollections and future trends. Biochim.
Biophys. Acta 1780, 325–346 (2008).
n Recent review comprising a nice
historical excursus.
19 Haltiwanger RS, Lowe JB: Role of
glycosylation in development. Annu. Rev.
Biochem. 73, 491–537 (2004).
20 Cobb BA, Kasper DL: Coming of age:
carbohydrates and immunity. Eur.
J. Immunol. 35, 352–356 (2005).
21 Carbohydrate Drug Design. Klyosov AA,
Witczak ZJ, Platt D (Eds). ACS, Washington,
DC, USA (2006).
22 Ernst B, Magnani JL: From carbohydrate
leads to glycomimetic drugs. Nat. Rev. Drug
Discov. 8, 661–677 (2009).
State of the art concerning rational design
n

neuroscience. Progr. Neurobiol. 87, 133–170 of glycomimetics.

anti-tumor activity. Biomaterials 27, 119–126
(2009).
(2006). 23 Verez-Bencomo V, Fernández-Santana V,
7 Dobrovolskaia MA, Germolec DR, Hardy E et al.: A synthetic conjugate
15 Liu Z, Jiao Y, Wang Y, Zhou C, Zhang Z:
Weaver JL: Evaluation of nanoparticle polysaccharide vaccine against Haemophilus
Polysaccharides-based nanoparticles as drug
immunotoxicity. Nat. Nanotechnol. 4, influenzae type b. Science 305, 522–525
delivery systems. Adv. Drug Deliv. Rev. 60,
411–414 (2009). (2004).
1650–1662 (2008).

790 Nanomedicine (2010) 5(5) future science group

 Glyconanoparticles: multifunctional nanomaterials for biomedical applications
24 Carbohydrate-Based Vaccines and
Immunotherapies. Guo Z, Boons G-J (Eds).
Wiley-VCH Verlag GmbH & Co. KGaA,
Weinheim, Germany (2009).
25 Larsen K, Thygesen MB, Guillaumie F,
Willats WGT, Jensen KJ: Solid-phase
chemicals tools for glycobiology. Carbohydr.
Res. 341, 1209–1234 (2006).
26 Chabre YM, Roy R: Design and creativity in
synthesis of multivalent neoglycoconjugates.
Adv. Carbohydr. Chem. Biochem. 63, 165–393
(2010).
n A meticulous account about different
strategies leading to a vast range of
multivalent glycoconjugate architectures.
27 de la Fuente JM, Penadés S:
Glyconanoparticles: types, synthesis and
applications in glycoscience, biomedicine and
material science. Biochim. Biophys. Acta Gen.
Subj. 1760, 636–651 (2006).
28 de la Fuente JM, Penadés S: Understanding
carbohydrate–carbohydrate interactions by
means of glyconanotechnology. Glycoconj. J.
21, 149–163, (2004).
29 Marradi M, Martín-Lomas M, Penadés S:
Glyconanoparticles: polyvalent tools to study
carbohydrate-based interactions. Adv.
Carbohydr. Chem. Biochem. (2010) (In Press).
30 Eggens I, Fenderson B, Toyokuni T, Dean B,
Stroud M, Hakomori S-I: Specific interaction
between Lex and Lex determinants – a possible
basis for cell recognition in preimplantation
embryos and in embryonal carcinoma-cells.
J. Biol. Chem. 264, 9476–9484 (1989).
31 de la Fuente JM, Barrientos AG, Rojas TC
et al.: Gold glyconanoparticles as water-
soluble polyvalent models to study
carbohydrate interactions. Angew. Chem.
Int. Ed. 40, 2257 (2001).
32 Barrientos AG, de la Fuente JM, Rojas TC ,
Fernández A, Penadés S: Gold
glyconanoparticles: synthetic polyvalent
ligands mimicking glycocalyx-like surfaces as
tools for glycobiological studies. Chem. Eur. J.
9, 1909–1921 (2003).
33 Ojeda R, de Paz JL, Barrientos AG,
Martín-Lomas M, Penadés S: Preparation of
multifunctional glyconanoparticles as a
platform for potential carbohydrate-based
anticancer vaccines. Carbohydr. Res. 342,
448–459 (2007).
34 Mammen M, Choi S-K, Whitesides GM:
Polyvalent interactions in biological systems:
implications for design and use of multivalent
ligands and inhibitors. Angew. Chem. Int. Ed.
Engl. 37, 2755–2794 (1998).
35 Hakomori S: Carbohydrate–carbohydrate
interaction as an initial step in cell
recognition. Pure Appl. Chem. 63, 473–482
(1991).
future science group
36 Rojo J, Diaz V, de la Fuente JM et al.: Gold
glyconanoparticles as new tools in
antiadhesive therapy. ChemBioChem 5,
291–297 (2004).
37 Hakomori S-I, Zhang Y: Glycosphingolipid
antigens and cancer therapy. Chem. Biol. 4,
97–104 (1997).
38 McEver RP: Selectin–carbohydrate
interactions during inflammation and
metastasis. Glycoconj. J. 14, 585–591
(1997).
39 Hakomori S-I: The glycosynapse. Proc. Natl
Acad. Sci. USA 99, 225–232 (2002).
40 Barré-Sinoussi F: HIV: a discovery opening
the road to novel scientific knowledge and
global health improvement. Angew. Chem.
Int. Ed. 48, 5809–5814 (2009).
41 Lue J, Hsu M, Yang D, Marx P, Chen Z,
Cheng-Mayer C: Addition of a single gp120
glycan confers increased binding to dendritic
cell-specific ICAM-3-grabbing nonintegrin
and neutralization escape to HIV type 1.
J. Virol. 76, 10299–10306 (2002).
42 Reitter JN, Means RE, Desrosiers RC: A role
for carbohydrates in immune evasion in
AIDS. Nat. Med. 4, 679–684 (1998).
43 Nolting B, Yu J-J, Liu G-Y, Cho S-J,
Kauzlarich S, Gervay-Hague J: Synthesis of
gold glyconanoparticles and biological
evaluation of recombinant gp120 interactions.
Langmuir 19, 6465–6473 (2003).
44 van Kooyk Y, Geijtenbeek TBH: DC-SIGN:
escape mechanism for pathogens. Nat. Rev.
Immunol. 3, 697–709 (2003).
45 Geijtenbeek TBH, Kwon DS, Torensma R
et al.: DC-SIGN, a dendritic cell-specific
HIV-1-binding protein that enhances
trans-infection of T cells. Cell 100, 587–597
(2000).
46 Martínez-Ávila O, Hijazi K, Marradi M et al.:
Gold manno-glyconanoparticles: multivalent
systems to block HIV-1 gp120 binding to the
lectin DC-SIGN. Chem. Eur. J. 15,
9874–9888 (2009).
47 Martínez-Ávila O, Bedoya LM, Marradi M,
Clavel C, Alcamí J, Penadés S: Multivalent
manno-glyconanoparticles inhibit DC-SIGN-
mediated HIV-1 trans-infection of human
T cells. ChemBioChem 10, 1806–1809
(2009).
48 Lin C-C, Yeh Y-C, Yang C-Y et al.: Selective
binding of mannose-encapsulated gold
nanoparticles to type 1 pili in Escherichia coli.
J. Am. Chem. Soc. 124, 3508–3509 (2002).
49 El-Boubbou K, Gruden C, Huang X:
Magnetic glyco-nanoparticles: a unique tool
for rapid pathogen detection,
decontamination, and strain differentiation.
J. Am. Chem. Soc. 129, 13392–13393
(2007).
www.futuremedicine.com
Review
50 Luo PG, Stutzenberger FJ: Nanotechnology
in the detection and control of
microorganisms. Adv. Appl. Microbiol. 63,
145–181 (2008).
51 Panáček A, Kvítek L, Prucek R et al.: Silver
colloid nanoparticles: synthesis,
characterization, and their antibacterial
activity. J. Phys. Chem. B 110, 16248–16253
(2006).
52 Qi L, Xu Z, Jiang X, Hu C, Zou X:
Preparation and antibacterial activity of
chitosan nanoparticles. Carbohydr. Res. 339,
2693–2700 (2004).
53 Shi Z, Neoh KG, Kanga ET, Wang W:
Antibacterial and mechanical properties of
bone cement impregnated with chitosan
nanoparticles. Biomaterials 27, 2440–2449
(2006).
54 Wei D, Sun W, Qian W, Ye Y, Ma X:
The synthesis of chitosan-based silver
nanoparticles and their antibacterial
activity. Carbohydr. Res. 344, 2375–2382
(2009).
55 de la Fuente JM, Alcántara D, Eaton P et al.:
Gold and gold-iron oxide magnetic
glyconanoparticles: synthesis, characterization
and magnetic properties. J. Phys. Chem. B
110, 13021–13028 (2006).
56 van Kasteren SI, Campbell SJ, Serres S,
Anthony DC, Sibson NR, Davis BG:
Glyconanoparticles allow pre-symptomatic
in vivo imaging of brain disease. Proc. Natl
Acad. Sci. USA 106, 18–23 (2009).
n First example of construction and in vivo
application of highly multivalent and
MRI-visible magnetic glyconanoparticles
(GNPs) for direct detection of endothelial
markers E-/P-selectin in acute inflammation.
57 Marradi M, Alcántara D, de la Fuente JM,
García-Martín ML, Cerdán S, Penadés S:
Paramagnetic Gd-based gold
glyconanoparticles as probes for MRI: tuning
relaxivities with sugars. Chem. Commun.
3922–3924 (2009).
58 El-Boubbou K, Zhu DC, Vasileiou C et al.:
Magnetic glyco-nanoparticles: a tool to
detect, differentiate, and unlock the
glyco-codes of cancer via magnetic resonance
imaging. J. Am. Chem. Soc. 132, 4490–4499
(2010).
n Detection and selective differentiation
of cancer cells in vitro by means of simple
sugar-based magnetic GNPs and
MRI techniques.
59 Tosi G, Costantino L, Rivasi F et al.:
Targeting the central nervous system: in vivo
experiments with peptide-derivatized
nanoparticles loaded with loperamide and
rhodamine-123. J. Control. Rel. 122, 1–9
(2007).
791
Review García, Marradi & Penadés
60 Veiseh O, Sun C, Fang C et al.: Specific
targeting of brain tumors with an optical/
magnetic resonance imaging nanoprobe
across the blood–brain barrier. Cancer Res. 69,
6200–6207 (2009).
61 Lee H, Lee K, Kim IK, Park TG: Synthesis,
characterization, and in vivo diagnostic
applications of hyaluronic acid immobilized
gold nanoprobes. Biomaterials 29, 4709–4718
(2008).
n Interesting example of in vivo fluorescent
imaging of diseases with gold GNPs
responsive to pathological stimuli.
62 Kikkeri R, Lepenies B, Adibekian A,
Laurino P, Seeberger PH: In vitro imaging
and in vivo liver targeting with carbohydrate
capped quantum dots. J. Am. Chem. Soc. 131,
2110–2112 (2009).
63 Ban Z, Bosques CJ, Sasisekharan R: A simple
assay to probe disease-associated enzyme
activity using glycosaminoglycan-assisted
synthesized gold nanoparticles. Org. Biomol.
Chem. 6, 4290–4292 (2008).
64 Chikae M, Fukuda T, Kerman K, Idegami K,
Miura Y, Tamiya E: Amyloid-b detection
with saccharide immobilized gold
nanoparticle on carbon electrode.
Bioelectrochemistry 74, 118–123 (2008).
65 La Belle JT, Gerlach JQ, Svarovsky S, Joshi L:
Label-free impedimetric detention of glycan–
lectin interactions. Anal. Chem. 79,
6959–6964 (2007).
66 Hearty S, Leonard P, O’Kennedy R: Barcodes
check out prostate cancer. Nat. Nanotechnol.
5, 9–10 (2010).
792
67 Murphy CJ, Gole AM, Stone JW et al.: Gold
nanoparticles in biology: beyond toxicity to
cellular imaging. Acc. Chem. Res. 41,
1721–1730 (2008).
68 Nanotechnologies For The Life Sciences
(Volume 5, Nanomaterials – Toxicity,  
Health And Environmental Issues). Kumar
CSSR (Ed.). Wiley-VCH Verlag GmbH
& Co. KGaA, Weinheim, Germany
(2006).
69 de la Fuente JM, Alcantara D, Penadés S:
Cell response to magnetic glyconanoparticles:
does the carbohydrate matter? IEEE Trans.
Biosci. 6, 275–281 (2007).
70 Maysinger D, Lovrić J, Eisenberg A, Savić R:
Fate of micelles and quantum dots in cells.
Eur. J. Pharm. Biopharm. 65, 270–281
(2007).
71 Smith AM, Duan H, Mohs AM,
Nie S: Bioconjugated quantum dots for
in vivo molecular and cellular imaging.
Adv. Drug Deliv. Rev. 60, 1226–1240
(2008).
72 Cambi A, Lidke DS, Arndt-Jovin DJ,
Figdor CG, Jovin TM: Ligand-conjugated
quantum dots monitor antigen uptake and
processing by dendritic cells. Nano Lett. 7,
970–977 (2007).
73 Jiang X, Ahmed M, Deng Z, Narain R:
Biotinylated glyco-functionalized quantum
dots: synthesis, characterization, and
cytotoxicity studies. Bioconj. Chem. 20,
994–1001 (2009).
74 Marnett LJ: Nanotoxicology – a new
frontier. Chem. Res. Toxicol. 22, 1491
(2009).
Nanomedicine (2010) 5(5)
75 Fent GM, Casteel SW, Kim DY et al.:
Biodistribution of maltose and gum arabic
hybrid gold nanoparticles after intravenous
injection in juvenile swine. Nanomed.
Nanotechnol. 5, 128–135 (2009).
„„Websites
101 The ETP Nanomedicine is an initiative led by
industry and set up together with the
European Commission.
www.etp-nanomedicine.eu/public
102 Nanomedicine is an ongoing research
program supported by the NIH
Common Fund.
http://nihroadmap.nih.gov/nanomedicine
103 GENNESYS is the acronym of Grand
European Initiative on Nanoscience and
Nanotechnology using Neutron and
Synchrotron Radiation Sources.
www.mf.mpg.de/mpg/
websiteMetallforschung/english/
veroeffentlichungen/GENNESYS/
104 Smith & Nephew sells and distributes
Acticoat™ with SILCRYST™ nanocrystals
under a licensing agreement with NUCRYST
Pharmaceuticals Corporation.
www.acticoat.com
105 Mantovani E, Procari A, Robinson DKR,
Morrison MJ, Geertsma RE: Development in
nanotechnology regulation and standards –
report of the Observatory Nano. May 2009.
www.observatorynano.eu
Comprehensive overview of the current
n
international situation in the field of
nanotechnology regulation.
future science group