Journal of Drug Delivery Science and Technology xxx (2015) 1e12

Contents lists available at ScienceDirect

Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Invited paper

From macro to nano polysaccharide hydrogels: An opportunity for

the delivery of drugs
Franco Alhaique*, Maria Antonietta Casadei, Claudia Cencetti, Tommasina Coviello,
Chiara Di Meo, Pietro Matricardi, Elita Montanari, Settimio Pacelli, Patrizia Paolicelli
Department of Drug Chemistry and Technologies, “Sapienza” University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: This overview follows the evolution of the studies carried out, mainly during last years, on poly-
Received 30 July 2015 saccharide hydrogels, with particular attention on the researches carried out in our department, often in
Received in revised form collaboration with other groups in our country and abroad. The review points out the importance of this
24 September 2015
type of networks for the optimization of drug delivery and targeting in the various forms of macro, micro
Accepted 24 September 2015
and nano systems. It is also shown that these materials are suitable for the culture of different types of
Available online xxx
cells. Release mechanisms are reported and explained by different physico-chemical approaches and by
means of molecular dynamics simulations and the anomalous swelling behavior of a scleroglucan/borax
Keywords:
Polysaccharides
hydrogel is thoroughly discussed. The role of polymer combinations forming interpenetrated structures
Hydrogels is explained in terms of specific properties which significantly differ from those of the constituent
Interpenetrated polymer networks polymers, thus allowing appropriate tailoring of the delivery rates. Finally the wide possibilities of ap-
Nanohydrogels plications of nanogel structures which allow combination therapies for cancer treatment and can be
Drug delivery suitable for intracellular targeting are reported. The studies on polysaccharide hydrogels are still in
Targeting progress and it is underlined that future researches, more focused on the passage from lab to market,
should be further stimulated.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction interest. Furthermore, it is worth to point out how, throughout the

Together with the identification of new bioactive compounds,
the release of drugs from appropriate dosage forms at prefixed time
intervals, and at predetermined rates, as well as the targeting to
specific sites represent the main challenges for scientists involved
in pharmaceutical studies. It can be stated that a scientific approach
to modified drug delivery studies dates back to the mid '60s [1] and
in recent years significant advances, with an exponential trend of
publications, have been achieved in the area of controlled/modified
release. Nevertheless, much work is still needed to further improve
the treatment of clinical pathologies. At the same time, the rapid
evolution of tissue engineering stimulated innovative studies on
biocompatible materials suitable for the culture of different types of
cells. For all these purposes synthetic and natural polymers have
been used and among them natural polysaccharides, and their
derivatives, represent a class of macromolecules of particular
years, three different periods can be identified which can be
respectively called: macro-, micro- and nano-ages [2].
A similar trend was followed by the research team at the Faculty
of Pharmacy at “Sapienza” University of Rome, often in collabora-
tion with colleagues in Italy and abroad. Actually the studies on
drug delivery carried out in our labs began in 1981 with one of the
first published paper on smart polymers [3], while those related to
the use of polysaccharides, our most studied polymers, started
eight years later [4].
Anyhow, in this review we intend to give, within an interna-
tional frame, an overview on researches, mainly recent, related to
macro- micro- and nano-structures, with particular attention to
hydrogels, a topic that will be discussed in more detail in the sec-
ond part of this paper. Future perspectives of this type of research
activities will also be given.
2. Why polysaccharides?

* Corresponding author. Among the wide variety of macromolecules that are actually
E-mail address: franco.alhaique@uniroma1.it (F. Alhaique). used, or have been studied, for the formulation of conventional and

http://dx.doi.org/10.1016/j.jddst.2015.09.018
1773-2247/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
2 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12
modified release dosage forms, natural polysaccharides and their
derivatives are indeed the most employed and versatile polymeric
materials. It must be also pointed out that polysaccharides show an
even wider variety of biomedical applications, ranging from their
intrinsic biological activity to their ability in living cell encapsula-
tion, from bone and cartilage repair to the preparation of friendly
scaffolds for tissue engineering. Furthermore, looking at quite close
areas of interest, polysaccharides are very often used as food ad-
ditives and are also present in cosmetic formulations and personal
care products. The peculiar and diversified properties of these
macromolecules can be related to the different molecular weights
(as well as their distributions) and chemical composition that these
polymers may have and also to the presence of a large number of
reactive groups. These properties allow numerous chemical mod-
ifications that can be appropriately tailored according to the spe-
cific use that is assigned to these multitasking materials.
Polysaccharides are usually abundant and available from various
renewable natural sources such as animals (e.g., chitosan, chon-
droitin), plants (e.g. pectin, guar gum, locust bean gum), algae (e.g.
alginates, agar, carrageenans), microbes and fungi (e.g. dextran,
xanthan gum, gellan, scleroglucan). Finally, but not less important,
polysaccharides are, with only few exceptions, biocompatible and
non-toxic products and can be classified as GRAS (Generally
Recognized as Safe).
3. An overview on some polysaccharide hydrogels suitable as
drug delivery matrices
Numerous polysaccharides are capable of forming gels in
appropriate conditions: alginate (Alg) and gellan show gelling
properties in aqueous solution in the presence of counterions
(physical network) [Scheme 1 A]; the synergistic interaction be-
tween Locust Bean Gum and Xanthan leads to gel formation; the
interaction of borax with the hydroxyl groups of polysaccharides
allows the formation of stable hydrogels and numerous bi/multi-
functional reagents behave as crosslinking agents for gel forma-
tion with many polymers (chemical network) [Scheme 1 B]. We
shall report here some examples of polysaccharides that have been
extensively studied as hydrogel matrices for drug delivery.
3.1. Scleroglucan
Among the numerous polysaccharides that have been proposed
for biomedical applications, Scleroglucan (Sclg) is undoubtedly a
particularly versatile polymer which is studied since many years,
both in its native form and after different types of chemical mod-
ifications [3,4]; and a quite recent review summarizes the most
important results obtained during the first twenty years [5].
Several approaches have been followed for the formation of
three-dimensional networks capable of swelling and suitable as
drug delivery systems. After the first studies based on chemical
crosslinking strategies, of particular interest and of peculiar
behavior is the gel formed with borax. This gel can be very easily
prepared by addition, to a Sclg solution, of an appropriate amount
(best: moles of borax ¼ moles of repeating unit of Sclg) of a borax
solution. For delivery experiments, the drug was preliminary dis-
solved in the polymer solution before the addition of borax, and the
obtained gel was freeze-dried and then compressed for tablet
preparation [6]. The release studies, carried out with model mole-
cules of different steric hindrance; i.e., Theophylline (TPH), Vitamin
B12 (VitB12) and Myoglobin (MGB), showed that, as expected,
delivery rates decreased as the van der Waals radius of the loaded
molecule increased, but, at the same time, a totally unexpected and
almost bizarre behavior was observed: when the tablets, obtained
from the Sclg/borax freeze-dried hydrogel, were soaked in a water
Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
medium an anisotropic swelling takes place, as it is possible to
visualize in Fig. 1, where the unidirectional increase of thickness
with respect to the original tablet can be observed (the color is due
to the presence of VitB12), while the increase of tablet diameter is
almost negligible.
Molecular dynamics simulations allowed shedding some light
on the anomalous swelling of this hydrogel. According to this
approach, an ordered configuration of parallel bundles of the Sclg
triple helices, hold together partially by covalent linkage and
partially by physical interactions with borate ions, was proposed
[7,8].
In Fig. 2 such structures, in the presence of the tested model
drugs, are reported. From these simulations it was also evidenced
that the ordered configuration is kept during swelling but is rapidly
lost in the absence of borax.
Furthermore, from the same Fig. 2 it can be evidenced that the
interaction between Sclg and borax leads to the formation of
nanochannels with different sizes, according to the steric hindrance
of the loaded molecule, thus allowing the diffusion and delivery
also of the larger tested molecule such as MGB. In this sense it is
also interesting to point out that the anisotropic effect above
described was more relevant in the case of the tablet prepared with
MGB because, in this case, a looser, but still ordered, structure is
formed (Fig. 2, c).
As a consequence of the anisotropic solvent uptake, also the
dynamo-mechanical and drug diffusion properties of the swelled
tablets were significantly different when detected along the two
directions (i.e., parallel and perpendicular with respect to the
compression force applied for the preparation of the tablets) [9,10].
More recently it has been evidenced, by means of NMR studies,
that the peculiar nanochannel structure together with the aniso-
tropic swelling induced also a difference in water diffusion. In fact,
for the first time in a polysaccharide hydrogel, a significantly
enhanced diffusion of water molecule (superdiffusive behavior)
was detected along the swelling direction [11].
Finally, it should be pointed out that all the reported behaviors
are peculiar and specific of that particular Sclg/borax hydrogel: in
fact, it was shown that while this polysaccharide is capable of
forming gels also with trivalent ions, such as Al (III) and Fe (III),
tablets obtained from these hydrogels never evidenced these type
of features [12].
3.2. Guar gum and locust bean gum
Just like Sclg, also Guar Gum (GG), both in its native form and
after chemical modifications, has been proposed, since numerous
years, for the formulation of modified release oral dosage forms,
and in particular for colon targeting [13,14].
More recently, GG was crosslinked with glutaraldehyde (GA)
and the release of guest molecules of different molecular weight
(TPH, VitB12, MGB), from tablets prepared by compression of the
freeze dried derivative, was evaluated and compared with that
from tablets obtained from the corresponding derivative of Sclg and
Locust Bean Gum as well as from the native polysaccharides.
Experimental data showed, quite unexpectedly, that, in the case
of the smaller molecules (TPH and VitB12), the presence in the
matrix of a well defined network, increased the delivery rate of the
guest molecules in comparison with the release profiles obtained
when no crosslinker was present. Actually, the introduction of a
spacer among the macromolecular chains, led to the formation of
meshes with sizes wide enough for a rather easy diffusion of small
molecules. On the other side, when the drugs diffuse only trough
the polymeric network, the chain entanglements hinder signifi-
cantly the free movement of the molecules, which showed an
appreciable decrease in rate of delivery [15].
 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12 3

Scheme 1. Hydrogel representation: A physical network, B chemical network, C semi-IPN and D IPN.

For a deeper investigation and a better understanding of the
delivery mechanism from the GA crosslinked GG network,
dynamo-mechanical and rheological characterizations were car-
ried out on the hydrogel system. In particular, the effect of tem-
perature and aging times on gel stability, were studied, together
with the critical exponents at the gel point and the fractal di-
mensions [16,17].
In spite of the relevant structural differences between Sclg
(triple helix and one of the most rigid polysaccharides) and GG
(flexible coil), an expected similarity between the two macromol-
ecules was evidenced when their borax networks were prepared.
Although in a lesser extent, also in the case of GG an anisotropic
elongation of the tablets, obtained from the freeze-dried hydrogel,
was observed and similarities were also detected from rheological
studies and by means of molecular dynamics simulations;
furthermore low field NMR investigations allowed a correlation
with the release studies performed with TPH and VitB12 as model
drugs [18,19].
It is interesting to point out that, when the MW of GG was
reduced by sonication, a corresponding reduction of the anisotropic
swelling behavior was detected [20].

3.3. Locust bean gum e xanthan

The two polysaccharides, Locust Bean Gum (LBG) and Xanthan

Fig. 1. The anisotropic swelling of a VitB12 - loaded tablet obtained from the Sclg/
borax freeze dried hydrogel. On the left the dry tablet; on the right the swelled tablet.
(Xanth), are both biocompatible and widely used, as single com-
ponents, in food industry as well as excipients in tablet formula-
tions, and are both non gelling components. Nevertheless, when
Xanth and LBG solutions are mixed together, a physical gel is
formed [21] which can be suitable also for pharmaceutical appli-
cations. In addition, the strength of the network depends on the
temperature preparation and on the weight ratio between the two
components [22]. In fact, by varying the relative amount of the two
polymers and taking into account the different conformation
assumed by the Xanth chains at room temperature (double-helix
ordered structure) and at T > 45  C (random coil conformation) it
was possible to modulate the mechanical properties of the gel.
Actually, when the polymer solutions were mixed, at a weight ratio
1:1 and in “hot conditions” (T  60  C) a strong network was ob-
tained (high degree of entanglements and physical crosslinks)
while, when the gel preparation occurred in “cold conditions”, the
formation of a weak gel was observed. Such difference is evidenced

Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
4 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12

Fig. 2. Molecular dynamics simulations of the Sclg/Borax hydrogel in the presence of TPH (a), VitB12 (b) and MGB(c). The arrows indicate some borate groups on the Sclg side-
chains that lead to the nanochannel formation. From Ref. [6] with permission.

also from the scanning electron micrographs reported in Fig. 3.
The versatility of Xanth/LBG matrices and the tailorable rheo-
logical properties, allowed to propose, for topical applications, a
formulation of these hydrogels containing curcumin [23].
Furthermore, taking into account that to overcome the problem
of the skin barrier the use of vesicular systems, such as liposomes
and niosomes (Nio), has been often proposed because of their
capability to enhance penetration across the skin, non ionic sur-
factant vesicles were loaded within this mixed polysaccharide gel
[24,25].
The system LBG/Xanth, loaded with Tween 20/cholesterol Nio
containing hydrophilic and lipophilic drugs (ibuprofen, caffeine
and ammonium glycyrrhizinate), showed that Nio were slowly
delivered, outside the hydrogel, as intact vesicles which were able
to act as carriers for enhanced dermal delivery. The innovative
system was thus able to combine the qualities of the polymeric gel
with those of non-ionic surfactant vesicles. Furthermore, shelf life
tests indicated that the gel formulation was stable also after a
period of one year, and rheological experiments showed that
dynamo-mechanical characteristics were similar to those of usual
topical formulations already in the market.
More recently, following a similar approach, a mixed system of
liposomes and a hydrogel obtained from another polysaccharide
(i.e., chitosan) was tested for delayed delivery of carboxyfluorescein
[26].
Finally, considering how critical can be the effect of the presence
of nanoparticles within the LBG/Xanth network, a wide study was
carried out about the influence that SiO2 nanoparticles loaded in
this hydrogel may have on the rheological performances of the
overall complex system [27].
4. IPN and semi-IPN based on polysaccharides for biomedical
applications
4.1. Semi-interpenetrating polymeric networks (semi-IPNs) and
interpenetrating polymeric networks (IPNs)
According to the IUPAC definition, an Interpenetrating Poly-
meric Network (IPN) is “a polymer comprising two or more net-
works which are at least partially interlaced on a molecular scale
but not covalently bonded to each other and cannot be separated
unless chemical bonds are broken. A mixture of two or more pre-
formed polymer networks is not an IPN”, while a semi-
Interpenetrating Polymeric Network (semi-IPN) is “a polymer
comprising one or more networks and one or more linear or
branched polymer(s) characterized by the penetration on a mo-
lecular scale of at least one of the networks by at least some of the
linear or branched macromolecules” (Scheme 1, C and D) [28].
In most cases these networks show physico-chemical properties
that can remarkably differ from those of the macromolecular con-
stituents. IPN and semi-IPN properties are very versatile because
they can be appropriately tailored according to the type of poly-
mers and their concentration, by the applied crosslinking method
as well as by the overall procedure used for their preparation;

Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12 5

Fig. 3. Scanning electron micrograph of surface morphology of freeze dried samples of Xanth/LBG prepared at 60 and 25  C and that of the two polysaccharides (magnification
800x). The relevant difference between the strong network obtained in “hot conditions” and the weak gel obtained in “cold conditions” is evidenced.

consequently these networks are suitable for a variety of applica-
tions, in particular in the field of biomedicine and for separation
processes [29e31].
Although studied for the first time more than one hundred years
ago [32], in recent years such multicomponent structures have
emerged as innovative biomaterials. Actually, depending on the
nature and the intrinsic properties of the components, the inter-
penetration of the polymer chains can result in the formation of
hydrogels which can be successfully used for biomedical and
pharmaceutical applications due to the combination of favorable
properties of each constituent polymer. In many cases, poly-
saccharides are selected for the formation of IPN hydrogel net-
works, which can be either chemically or physically crosslinked.
Sometimes both entangled macromolecules are based on poly-
saccharides, but often also combinations of synthetic polymers
together and polysaccharide chains are used to create (semi)-IPNs
for drug delivery and as scaffolds for cell cultures [33e41].
Quite a large number of polysaccharides has been investigated
for the design of semi-IPNs and IPNs for drug delivery and other
biomedical applications. In our labs, after a first approach on a
semi-IPN obtained with two polysaccharides (i.e., Sclg and Alg)
[42], other systems were prepared using mainly dextran (Dex), Alg
and hyaluronic acid (HA).
4.2. Dextran derivatives-alginate IPN hydrogel systems
Alg, produced by bacteria or extracted from marine brown algae,
is a well known polysaccharide composed of a polymer backbone of
1,4 linked b-D mannuronic acid (M) and a-L-guluronic acid (G)
residues. Alg contains homopolymeric M and G-blocks, that are
joined by regions of alternating structures [43,44].
The physico-chemical properties as well as the gelling ability of
Alg strongly depend on the M/G ratio and on the structure of the
alternating zones. Furthermore, it was evidenced that the G-blocks
are responsible for the so called “egg-box” gel structure formation
in the presence of divalent counterions (e.g. calcium, lead, and
copper).
The counterion-driven Alg gel formation occurs quite rapidly
and the obtained hydrogels have been studied for numerous
biomedical applications, such as drugs and proteins modified
release, in the form of films, microspheres, depot matrices, and, in
tissue engineering, as scaffolds for cartilage, bone and soft tissue
regeneration [45e49].
Because of its high versatility and tailorable mechanical prop-
erties, Alg has been used as one of the most studied building blocks
of interpenetrating polymer network.
Within this field of research we developed new polysaccharide
hydrogel IPN systems suitable for drug delivery and for tissue en-
gineering by blending Alg and Dex derivatives. The rationale behind
this study is the assumption that it is possible to modulate the Alg
hydrogel mechanical properties by interpenetrating, within its
network, a second polymer network which, in this case was a Dex
methacrylate (DexMa) derivative. Actually, the Dex hydrophobized
chains can disturb the formation of the Ca(II)-Alg hydrogel “egg-
boxes”, leading to a new semi-IPN hydrogel that can be injected
through a hypodermic needle. The obtained semi-IPN can then be
transformed into an IPN by UV photopolymerization of the double
bonds on the Dex chains, thus leading to a stable chemical network,
interpenetrated with the Alg-Ca(II) physical network, according to
the overall scheme reported in Fig. 4 A.
As evidenced by rheological measurements, using Dex with
different molecular weights, varying the polymer content and the
derivatization degree of Dex, and by UV irradiation for various in-
tervals of time it has been possible to modulate the mechanical
properties of the final IPN system; properties which, anyhow, al-
ways significantly differed from those of the original Alg physical
hydrogel. Correspondingly, the release rates of drugs loaded within
these networks were strictly dependent on the specific rheological
properties of the tested IPN and decreased by increasing the
crosslinking density of the DexMa network [50,51].
It must be underlined that the overall procedure used for the
semi-IPN and IPN hydrogel formation is very simple and does not
require hazardous or toxic chemicals, this being fundamental in the
case of materials suitable for biomedical applications. Furthermore,
the mild conditions adopted to form the IPN allow to load, within
the hydrogel, proteins/enzymes which can retain their activity, as
clearly indicated in Fig. 5 where the delivery profiles and the ac-
tivity of the released horseradish peroxidase enzyme (HRP) are

Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
6 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12

Fig. 4. A) Scheme adopted for the formation of the Alg-DexMa IPN. B) Scheme for an “in situ” polymerization of the syringeable semi-IPN. From Ref. [50] with modifications.

reported. depending on the specific composition, they were able to differ-

Summarizing: this system, in the form of a semi-IPN and after entiate, as it could be demonstrated by the deposition of collagen
addition of a selected drug/protein, can be injected in a specific type II [53] (Fig. 6).
target site and then photopolymerized in situ using an appropriate In order to further modulate the performances of the IPNs based
UV source, such as an UV optical fiber (Fig. 4 B). on Alg and dex-HEMA, and in particular to improve the bio-
By dropping aqueous solutions of Alg containing different elimination of the degradation products, the native Alg was
amounts of DexMa in a CaCl2 solution it was possible to obtain the
semi-IPN in the form of beads which, after UV irradiation in the
presence of Irgacure, gave the corresponding IPN network. The
beads average size, ranging from 2.0 to 2.5 mm, decreased as
DexMa concentration in the initial blend increased and their
entrapment efficiency of proteins such as BSA and HRP was relevant
(60 and 75% respectively). As expected a sustained release of the
proteins from the beads was obtained and the delivered HRP
maintained its enzymatic activity [52].
Although the above reported Alg/Dex-Ma interpenetrated
structure can be considered suitable for an in situ delivery system,
it cannot be concealed that it may show problems related to its
biodegradation; consequently hydroxyethyl-methacrylate-
derivatized dextran (dex-HEMA) was chosen for the formation of
the IPN with the Alg-Ca(II) physical hydrogel. Again different de-
grees of substitution (DS) and concentrations of dex-HEMA were
tested for an appropriate modulation of the mechanical properties
of the new system, and the delivery of a model protein (BSA) from
the various formulations was evaluated. In all cases the release was
essentially governed by a Fickian diffusion mechanism. For a better
characterization of this IPN, also swelling/degradation experiments
were carried out: the corresponding typical trend, in a HEPES
buffer, showed always an initial increase of weight of the tested
samples, due to solvent absorption, followed, after a maximum was
reached, by a decrease of weight related to the degradation of the
polymer network.
As expected, IPNs degradation was influenced by the dex-HEMA
DS and the polymer concentration. Complete degradation occurred
within 15e18 days for samples prepared with low DS values of dex-
HEMA, while IPNs with the highest DS degraded in 70e180 days,
thus indicating how versatile and tailorable can be this type of IPN.
Furthermore, taking into account the possibility to use this material
also for cell therapy purposes, encapsulation of expanded chon- Fig. 5. A) Release of HRP from the Alg-DexMa IPNs. B) percentage of enzymatic activity
drocytes in this IPN revealed that cells remained viable and, of released HRP with respect to the free enzyme. From Ref. [50] with modifications.

Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12 7

substituted with an Alg oxidized (oxAlg) with periodate to a dia-

ldehyde derivative that, being susceptible to hydrolytic scissions,
can be more easily eliminated from the human body. Although Alg
oxidation somehow affected the rheological properties of the
Ca(II)-Alg hydrogel, in combination with dex-HEMA it was possible
to obtain strong IPNs structures which were tested as matrices for
protein delivery (BSA and myoglobin) and were also degradable
after and initial swelling, as reported in Fig. 7 for different samples
obtained following this procedure [54].
Following the above reported approach of dropwise addition of
a mixed polymer solutions into the CaCl2 solution, oxAlg was used
also for the formation beads obtained by both physical and chem-
ical crosslinking of oxAlg and native Alg, where Alg was sometimes
partially substituted with Hyaluronic acid (HA). Swelling of such
beads significantly increased with increasing environmental pH
conditions thus allowing the authors to assert that such system has
“a great potential as drug delivery carrier” [55].
While in most of the above reported cases Dex derivatives were Fig. 7. Examples of swelling/degradation trend of some dex-HEMA-oxAlg IPNs. From
the interpenetrating second polymer, also other macromolecules Ref. 54 with permission.
were tested for semi-IPN and IPN formation. Actually, using HA
methacrylate derivatives and Alg, it was possible to obtain semi-
networks [57].
IPN hydrogels which were then cured using an UV source for the
A different, and somehow opposite approach, is based on the
IPN formation. The mechanical and release properties of this new
use of methacrylated Alg (Alg-Ma) as photocrosslinkable polymer
IPN system, based on two charged polysaccharides, was signifi-
which formed IPNs with gelatin. The obtained material exhibited
cantly affected by the environmental ionic strength: the shield ef-
no cytotoxic effects and excellent cytocompatibility and the MTT
fect of an added salt induced a higher flexibility to the chains,
assay confirmed that Fibroblasts L929 cells readily proliferated
leading to a more interpenetrated system with improved elasticity.
when seeded on it [58].
Furthermore, the obtained material was suitable for sustained
enzyme delivery (HRP) which maintained its activity [56].
For a better understanding of the release behavior of several Alg/ 4.3. IPNs based on other polysaccharides
Dex-Ma IPNs, by means of a combination of different techniques
(i.e., rheology, low field NMR and cryoporosimetry) it also was Apart from Alg, other polysaccharides were investigated for IPN
possible to evaluate the mesh size distribution of the studied formation. Dex-Ma was used for the preparation of IPNs with Sclg

Fig. 6. Up: Cell viability assay (stains live cells green and dead cells red), of A3D108 (A) and A3D1016 (B) chondrocyte-seeded IPN hydrogels after 4 days of in vitro culture. Down:
Safranin O staining (A, B, and C; stains proteoglycans red) and immunolocalization of chondrogenic marker collagen type II (D, E, and F; brown) of A3D108 (A and D), A3D1012 (B
and E) and A3D1016 (C and F) chondrocyte-seeded IPN hydrogels after 4 weeks of in vitro culture. Scale bars represents 100 mm. From Ref. [53] with modifications. (For inter-
pretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
8 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12
and its carboxymethyl derivative (Sclg-CM). Due to a synergistic
interaction with Dex-Ma, the functionalized Sclg gave networks
with a higher viscosity than those prepared with the native Sclg. All
IPN hydrogels obtained with these polymers were able to swell
when in contact with biological fluids and, in the case of Sclg-CM, a
pH dependence was observed, with higher water uptake at pH 7.4
because of the electrostatic repulsion among the chains; such effect
was remarkably reduced in the presence of salts acting as shields of
the charges present on the macromolecules. When tested as drug
delivery matrices, the systems showed release rates inversely
related to the steric hindrance of the loaded model drug: i.e. a rapid
release of a small molecule such as theophylline and a prolonged
release of Vitamin B12 (Vit.B12); while, in the case of a protein
(myoglobin), the IPN behaved almost as depot systems (only
30e40% released after 350 h) [59].
Several IPNs were also prepared with Gellan Gum (Ge) which,
like Alg, is capable to form physical hydrogels with divalent ions;
but only few of them have been proposed for biomedical applica-
tions. Ge was coupled with agarose and the physico-chemical
properties of the obtained materials were investigated [60].
While an IPN Ge/Albumin IPN, in the form of microcapsules
entrapped with diltiazemeresin complex, was proposed as
controlled delivery system [61].
More recently novel photochemically linked IPN hydrogels were
prepared with Ge methacrylate (Ge-MA) and polyethylene glycol
dimethacrylate (PEG-Dma). The combination of the two polymeric
solutions gave a system which can be easily injected and then
photocrosslinked in situ. The properties of the obtained network
were deeply influenced by the concentration and molecular weight
of PEG-Dma and its cytocompatibility was evidenced. When tested
as a delivery system, the release rate was, as expected, faster for
small drug molecules, such as sulindac, than for larger ones (e.g.
Vit.B12) [62].
It has been reported that a particular type of IPN is represented
by the Double Network hydrogel (DN) which differs from the
conventional interpenetrating systems, due to the existence, in the
same structure of two networks in sharp contrast in terms of ri-
gidity, molecular weight and cross-linking density. A DN hydrogel is
commonly obtained following a two-step procedure. The first one
concerns the preparation of a stiff and brittle gel of a poly-
electrolyte, which is then immersed in an aqueous solution of a
second monomer. After the diffusion, the monomer is crosslinked
leading to the formation of a second, soft and ductile network in-
side the gel. This kind of system shows high toughness due to a
synergistic effect of the binary asymmetric structure, rather than to
a linear combination of the two component networks, as it occurs
in the conventional IPN [63].
Following this suggestion, both the conventional IPN strategy
and the DN strategy were applied on two polymeric systems based
on Ge and PEG-DMA where the second polymer represented the
diffusing species within the Ca(II)-Ge hydrogel. The DN approach
gave a material which showed improved mechanical properties
with respect to that obtained by means of the usual IPN strategy. In
all cases the release rate of model drugs was deeply influenced by
the molecular weight of PEG-DMA used for the formation of the
complex network. Finally, beside the improved mechanical prop-
erties, the new hydrogels showed a good biocompatibility when
tested in vitro on a human fibroblast cell line [64].
5. Polysaccharide nanohydrogels
In recent years, several self-assembling polymeric nanoparticles
or nanohydrogels (NHs) have been developed. Such nano-
structures can be easily prepared and show potential applications
as drug vehicles which, in most cases, are biocompatible and/or
Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
biodegradable, depending on the nature of the used polymer
[65,66].
The term nanohydrogel (or nanogel) indicates a particular type
of nanoparticles having a size between 10 and 1000 nm together
with the structure and the characteristics of hydrogels, such as high
flexibility, versatility, water absorption, high biocompatibility.
Because of their properties, nanogels are gaining considerable
importance for applications in the field of pharmaceutics as drug
carriers for parenteral (iv, im, sc), inhalatory or topical
administration.
In particular, it has been observed that a hydrophilic polymer,
such as a polysaccharide, when appropriately functionalized with
hydrophobic moieties and exposed to particular conditions in an
aqueous environment, can lead to self-assembled structures with
nanohydrogel characteristics [67].
At present, several different methods for the synthesis of func-
tionalized polysaccharides suitable for the preparation of nano-
hydrogels are known; among them the most widely applied, since
several years, is the derivatization of the polymer chains with
cholesterol (Chol) derivatives or cholanic acid [68e71]. Such mol-
ecules, when linked to the polysaccharide chains, give to the system
the needed amphiphilic properties which allow, after appropriate
treatments (e.g. sonication), the self-assembling process in water
and/or physiological solutions.
The obtained self-assembled nanogels exhibit structural fea-
tures that make them carriers suitable for both hydrophilic and
hydrophobic molecules, that can be loaded, within the nanogel,
each one in the respective domain. (Fig. 8).
The best performances are actually obtained by loading hydro-
phobic drugs, which exhibit a better permanence inside the
nanogels, with an extremely slow release in storage conditions.In
our laboratories our attention has been mainly focused on the two
natural polysaccharides Ge and HA which, as above reported were
also investigated for IPN formation. Ge is of microbial origin and is
formed by a tetrasaccharide repeating unit; while HA, of animal
origin, is composed by units of glucuronic acid and N-acetylglu-
cosamine. Both polysaccharides exhibit unique features, such as
biocompatibility, mucoadhesion, non-immunogenicity, which
make them suitable for the formation of nanogels; moreover, Ge
shows high gelling properties, while HA shows a high bioactivity, in
particular a selective interaction with several cell receptors, the
most important being the CD44 (Clusters of Differentiation, CD).
CD44 mediates the internalization of HA into the cell via endocy-
tosis and the subsequent degradation within the lysosomes by
means of hyaluronidases; an important feature of CD44 is to be a
receptor overexpressed in various isoforms of several tumor cells,
particularly in human solid tumors [72].
Following these concepts, several NHs based on the polymeric
derivatives GeePred (prednisolone), Ge-Chol [73], HA-Chol [74,75]
and HA-Rfv (riboflavin) [76] were prepared, optimizing with time
the synthesis, the preparations and the formulations, and also
performing a detailed physico-chemical study of these materials for
a better understanding of their behavior as drug carriers. Ge was
previously depolymerized by probe sonication, in order to obtain a
polymer with reduced molecular weight, better suitable for the
nanostructure formation [77].
Hydrophobic moieties were then linked to the polysaccharide
chains by a double-step reaction using appropriate bifunctional
linkers, such as 4-Br-butyric acid or 1,6-dibromohexane, and the
polymers were recovered as freeze-dried products.
Several NHs formation techniques were investigated, along with
different drug-loading methods; in particular ultrasonication,
nanoprecipitation, loading from preformed drug film, co-
nanoprepitation or loading from aqueous solvent into preformed
NHs; moreover, an innovative procedure that allowed, in a single
 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12 9

Fig. 8. Schematic representation of the formation of nanogels from a hydrophobized polysaccharide and of the drug loading process. The different localization, within the nanogel,
of a hydrophilic drug and a hydrophobic drug is evidenced.

step carried out in autoclave, the simultaneous formation, sterili-
zation and drug loading of the nanogel, was developed and
patented [78].
Depending on the polymer platform and formation method,
NHs ranging from 150 to 350 nm were obtained, in all cases with
low polydispersity index (PDI < 0.15e0.20). CRYO-TEM micro-
graphs showed that NHs have a spherical shape and a homoge-
neous structure (Fig. 9).
The high negative z-potential values detected for all tested NHs
(about 30/40 mV) were capable of preventing aggregation
phenomena; furthermore, these nanostructures were extremely
stable in water at different temperatures and also resistant at
lyophilization conditions in the presence of a cryoprotectant such
as dextrose. The freeze-dried products, when re-suspended in
water, re-formed NHs that retained dimensions, z-potential and the
other physico-chemical features of the original ones.
Together with the optimization of NHs preparation and their
characterization, the obtained systems were tested as carriers of
antitumoral, antiinflammatory and antibiotic drugs.
Ge -based nanohydrogels were studied for the treatment of
cancer cells by carrying an anticancer drug (paclitaxel, PCT)
together with the steroid prednisolone (Pred) for a combined
therapy [79].
For this purpose, Pred was chemically bound to the polymer
while the anticancer drug was loaded within the network by means
of the solvent casting technique. The final nanogel suspension
contained 140 mg/mL of loaded PCT: a relevant result considering
the high hydrophobicity and the particular low solubility of the
drug (less than 0.1 mg/mL in water). It has been suggested that PCT
was loaded within the hydrophobic domains formed by the inter-
action with Pred. The system, which was stable over one week, was
tested on several tumor cell lines, showing a significant advantage
when compared to the free PCT, administered in DMSO. Obtained
data confirmed that the combination of PCT administered in
nanogels with Pred was more effective than the free drug, espe-
cially at low concentrations and even on cancer cell lines less
sensitive to PCT. In particular, ovarian cancer Skov-3, which are
resistant to PCT, were responsive to the treatment with the novel
nanogels. Furthermore, a reduction of antitumor activity was
detected when, for NH formation, Chol, instead of Pred, was used as
inactive steroidal reference, thus indicating the actual and funda-
mental importance of a combined antitumor-antiinflammatory
therapy (Fig. 10).
Taking into account that most antimicrobials are not capable of
diffusing through cellular membranes while bacteria are often able
to penetrate inside host cells, innovative self-assembled NHs have
been studied as drug carriers for intracellular compartment tar-
geting. The system consisted in HA-Chol NHs loaded with levo-
floxacin (LVF), one of the most widely used fluoroquinolone
antibiotics. This new DDS, was specifically designed to increase the
cellular uptake of the drug thus improving its efficacy in the
treatment of intracellular infections caused, for instance, by

Fig. 9. Cryo-TEM micrograph of A) HA-Chol nanogel, B) HA-Rfv nanogel.

Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
10 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12
Fig. 10. In vitro viability of Skov-3 cancer cells after incubation with free PCT, PCT-
loaded. Ge-Chol NHs and PCT-loaded Ge-Pred NHs. Values are mean ± SD (n ¼ 3).
From Ref. [79] with permission.
Staphylococcus aureus and Pseudomonas aeruginosa [80]. LVF
loading within NHs was carried out by means of the co-
precipitation method and the obtained networks were so stable
that autoclaving sterilization and freeze drying, in the presence of
dextrose, could be carried out without significant variations of the
original structure.
The MIC values of NHs-LVF for S. aureus and P. aeruginosa
compared with those of free LVF showed that, when entrapped
within NHs, the drug retains its efficacy against these bacteria;
furthermore, experiments carried out on HeLa epithelial cells
infected with the same bacteria, demonstrated that LVF within NHs,
showed a higher intracellular activity compared to that of free LVF
(Fig. 11), thus confirming that by loading the LVF in HA-Chol NHs, it
is possible to increase the antibiotic cellular uptake and promote
the levofloxacin therapeutic efficacy against intracellular
infections.
As previously pointed out, polysaccharide hydrogels usually
show good biocompatibility and their high water content makes
them suitable for protein/enzyme loading and delivery; conse-
quently also NHs based on polysaccharides have been tested for
Fig. 11. Activity of LVF and LVF-NHs (both freshly prepared and resuspended following
freeze drying) on intracellular P. aeruginosa (PAO-1) and S. aureus (USA 300-0114).
From this figure it can be observed that LVF loading in HA-Chol NHs leads to an in-
crease of cellular uptake of the antibiotic and consequently to its therapeutic efficacy
against intracellular infections. From. Ref. [80] with permission.
Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
protein immobilization and vehiculation. With the aim of
improving a proposed therapy against melanoma cancer cells [81],
HA-Chol NHs. were tested as carriers for bovine serum albumin
oxidase (BSAO). For this purpose BSAO, an enzyme able to produce
anticancer species in situ, was covalently immobilized on HA-Chol
NHs by carbodiimide chemistry [74]. The activity of the immobi-
lized enzyme was checked in vitro (spermine assay) and on a model
melanoma cell line (M14 cells) and the obtained results indicated
that BSAO, covalently immobilized on NHs, retained its activity.
Further experiments, performed on human melanoma cancer cells,
demonstrated that, when melanoma cells were treated with the
immobilized enzyme, an increase of the cytotoxicity could be
observed, showing that these new HA-Chol NHs, suitable delivery
system for proteins, can represent an alternative strategy for the
melanoma treatment. Collected data suggest that self-assembled
NHs, based on hydrophobized Ge and HA chains, are suitable
nanovectors for drug and protein delivery for different therapeutic
purposes.
6. Conclusions
The polysaccharides, that we know today, are the result of the
long story of the evolution. Millions of years were necessary to
develop an incredible abundance of this type of macromolecules for
an extremely wide spectrum of functions (structural, biological,
biochemical, etc.).
Scientists, taking advantage of this long time activity of nature,
became, with time, more and more aware that it is possible to
exploit the properties of these materials for different applications,
and, among them, in the field of pharmaceutics and biomedicine.
This review on polysaccharide hydrogels, from “macro” to “nano”
systems, reports on some of the recent investigations carried out in
our laboratories and how they fit within the broad frame the
research activities in others countries related to this topic; it allows
also to understand how the polysaccharide hydrogel technology
significantly progressed during last decade, but, at the same time,
how these studies can be considered still at an infancy stage.
New appropriately tailored polysaccharide platforms are
needed for a better delivery modulation and targeting of drugs, in
particular those of the “last generation”, obtained from biotech-
nology and genetic engineering approaches, because of their
complex structure. Furthermore, deeper studies on combination
therapies are needed, together with applications in the field of
theranostics.
Finally, it should be observed that, although a relevant amount
of papers on polysaccharide hydrogels were published, at present,
only few attempts were made for the actual transfer of these re-
searches from lab to the market.
References
[1] J. Folkman, D.M. Long, The use of silicone rubber as a carrier for prolonged
drug therapy, J. Surg. Res. 4 (1964) 139e142.
[2] S. Yamane, A. Sugawara, A. Watanabe, K. Akiyoshi, Hybrid nanoapatite by
polysaccharide nanogel-templated mineralization, J. Bioact. Compat. Polym.
24 (2009) 151e168.
[3] F. Alhaique, M. Marchetti, F.M. Riccieri, E. Santucci, A polymeric film
responding in diffusion properties to environmental pH stimuli: a model for a
self regulating drug delivery system, J. Pharm. Pharmacol. 33 (1981) 413e418.
[4] E. Touitou, F. Alhaique, F.M. Riccieri, G. Riccioni, E. Santucci, Scleroglucan
sustained release oral preparations. Part I. In vitro experiments, Drug Des.
Deliv. 5 (1989) 141e148.
[5] T. Coviello, A. Palleschi, M. Grassi, P. Matricardi, G. Bocchinfuso, F. Alhaique,
Scleroglucan: a versatile polysaccharide for modified drug delivery, Molecules
10 (2005) 6e33.
[6] T. Coviello, M. Grassi, A. Palleschi, G. Bocchinfuso, G. Coluzzi, F. Banishoeib,
F. Alhaique, A new scleroglucan/borax hydrogel: swelling and drug release
studies, Int. J. Pharm. 289 (2005) 97e107.
[7] Palleschi, T. Coviello, G. Bocchinfuso, F. Alhaique, Investigation on a new on
 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12
sleroglucan/borax hydrogel: structure and drug release, Int. J. Pharm. 322
(2006) 13e21.
[8] G. Bocchinfuso, A. Palleschi, C. Mazzucca, T. Coviello, F. Alhaique, G. Marletta,
A theoretical and experimental study on a self-assembling polysaccharide
forming nanochannels: static and dynamic effects induced by a soft confine-
ment”, J. Phys. Chem. B 112 (2008) 6473e6483.
[9] T. Coviello, A. Palleschi, G. Bocchinfuso, L. Bertolo, P. Matricardi, F. Alhaique,
Peculiar behaviour of polysaccharide/borax hydrogel tablets: a dynamo-
mechanical characterization, Coll. Polym. Sci. 287 (2009) 413e423.
[10] M. Grassi, R. Lapasin, T. Coviello, P. Matricardi, C. Di Meo, F. Alhaique, Scle-
roglucan/borax/drug hydrogels: structure characterisation by means of
rheological and diffusion experiments, Carbohydr. Polym. 78 (2009) 377e383.
[11] C. Di Meo, T. Coviello, P. Matricardi, F. Alhaique, D. Capitani, R. La Manna,
Anysotropic enhanced water diffusion in scleroglucan tablets gels, Soft Matter
7 (2011) 6068e6075.
[12] T. Coviello, P. Matricardi, A. Balena, B. Chiapperino, F. Alhaique, Hydrogels
from scleroglucan and ionic crosslinkers: characterization and drug delivery,
J. Appl. Polym. Sci. 115 (2010) 3610e3622.
[13] A. Rubinstein, I. Gliko-Kabir, Synthesis and swelling-dependent enzymatic
degradation of borax-modified guar gum for colonic delivery purposes S.T.P,
Pharma Sci. 5 (1995) 41e46.
[14] I. Gliko-Kabir, B. Yagen, A. Penasi, A. Rubinstein, Low swelling crosslinked guar
gum and its potential use as colon specific drug carrier, Pharm. Res. 15 (1998)
1019e1025.
[15] T. Coviello, F. Alhaique, A. Dorigo, P. Matricardi, M. Grassi, Two gal-
actomannans and scleroglucan as matrices for drug delivery: preparation and
release studies, Eur. J. Pharm. Biopharm. 66 (2007) 200e209.
[16] C. Sandolo, P. Matricardi, F. Alhaique, T. Coviello, Dynamo-mechanical and
rheological characterization of guar gum hydrogels, Eur. Polym. J. 43 (2007)
3355e3367.
[17] C. Sandolo, P. Matricardi, F. Alhaique, T. Coviello, Effect of temperature and
cross-linking density on rheology of chemical cross-linked guar gum at the gel
point, Food Hydrocoll. 23 (2009) 210e220.
[18] G. Bocchinfuso, C. Mazzuca, C. Sandolo, S. Margheritelli, F. Alhaique,
T. Coviello, A. Palleschi, Guar gum and scleroglucan interactions with borax:
experimental and theoretical studies of an unexpected similarity, J. Phys.
Chem. B 114 (2010) 13059e13068.
[19] T. Coviello, P. Matricardi, F. Alhaique, R. Farra, G. Tesei, S. Fiorentino, F. Asaro,
G. Milcovich, M. Grassi, Guar Gum/Borax: rheological, low field NMR and
release characterization, eXPRESS Polym. Lett. 7 (2013) 733e746.
[20] S.A. Ansari, P. Matricardi, C. Cencetti, C. Di Meo, M. Carafa, C. Mazzuca,
A. Palleschi, D. Capitani, F. Alhaique, T. Coviello, Sonication-based improve-
ment of the physico-chemical properties of guar gum, as potential substrate
for modified drug delivery systems, BioMed Res. Int. (2013) 11. Article ID
985259.
[21] E.R. Morris, Mixed polymer gels. In P. Harris (Ed), Food Gels, pp. 291e359,
Elsevier, London U.K.
[22] C. Sandolo, D. Bulone, M.R. Mangione, S. Margheritelli, C. Di Meo, F. Alhaique,
P. Matricardi, Synregistic interaction of locust bean gum and xanthan inves-
tigated by rheology and light scattering, Carbohydr. Polym. 82 (2010)
733e741.
[23] E.J. Da_Lozzo, R.C.A. Moledo, C.D. Faraco, C.F. Ortolani-Machado,
T.M.B. Bresolin, J.L.M. Silveira, Curcumin/xanthan-galactomannan hydrogels:
rheological analysis and biocompatibility, Carbohydr. Polym. 93 (2013)
279e284.
[24] C. Marianecci, M. Carafa, L. Di Marzio, F. Rinaldi, C. Di Meo, F. Alhaique,
P. Matricardi, T. Coviello, A new vesicle-loaded hydrogel system suitable for
topical applications: preparation and characterization, J. Pharm. Pharm. Sci. 14
(2011) 336e346.
[25] T. Coviello, A.M. Trotta, C. Marianecci, M. Carafa, L. Di Marzio, Rinaldi, C. Di
Meo, F. Alhaique, P. Matricardi, Gel-embedded niosomes: preparation, char-
acterization and release studies of a new system for topical drug delivery, Coll.
Surf. B-Biointerf 125 (2015) 291e299.
[26] A. Billard, L. Pourchet, S. Malaise, P. Alcouffe, A. Montembault, C. Ladavie re,
Liposome-loaded chitosan physical hydrogel: toward a promising delayed-
release biosystem, Carbohydr. Polym. 115 (2015) 651e657.
[27] J.R.M. Kennedy, K.E. Kent, J.R. Brown, Rheology of dispersions of xanthan gum,
locust bean gum and mixed biopolymer gel with silicon dioxide nanoparticles,
Mater. Sci. Eng. C 48 (2015) 347e353.
[28] A.D. Jenkins, P. Kratochvìl, R.F.T. Stepto, U.W. Suter, Glossary of basic terms in
polymer science (IUPAC recommendations 1996), Pure Appl. Chem. 68 (1996)
2287e2311.
[29] L.M. Robeson, Polymer Blends: A Comprehensive Review, Hanser Publishers,
Munich, 2007. Hanser Gardner Publications, Cincinnati.
[30] P. Matricardi, C. Di Meo, T. Coviello, W.E. Hennink, F. Alhaique, Inter-
penetrating polymer networks polysaccharide hydrogels for drug delivery
and tissue engineering, Adv. Drug Deliv. Rev. 65 (2013) 1172e1187.
[31] E.S. Dragan, Design and applications of interpenetrating polymer network
hydrogels. A review, Chem. Eng. J. 243 (2014) 572e590.
[32] J.W. Aylsworth U.S. Pat. 1,111,284 (1914).
[33] Y.H. Bae, S.W. Kim, Hydrogel delivery systems based on polymer blends, block
co-polymers or interpenetrating networks, Adv. Drug Deliv. Rev. 11 (1993)
109e135.
[34] L.M. Robeson, Polymer Blends: A Comprehensive Review, Hanser Publishers,
Munich, 2007. Hanser Gardner Publications, Cincinnati.
Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
11
[35] Y.C. Liu, M.B. Park, Hydrogel based on interpenetrating polymer networks
based on poly(ethylene glycol) methyl ether acrylate and gelatine for vascular
tissue engineering, Biomaterials 30 (2009) 196e207.
[36] M.D. Brigham, A. Bick, E. Lo, A. Bendali, J.A. Burdick, A. Khademhosseini,
Mechanically robust and bioadhesive collagen and photocrosslinkable hyal-
uronic acid semi-interpenetrating networks, Tissue Eng. A 15 (2009)
1645e1653.
[37] M.D. Brigham, A. Bick, E. Lo, A. Bendali, J.A. Burdick, A. Khademhosseini,
Mechanically robust and bioadhesive collagen and photocrosslinkable hyal-
uronic acid semi-interpenetrating networks, Tissue Eng. A 15 (2009)
1645e1653.
[38] L. Weng, A. Gouldstone, Y. Wu, W. Chen, Mechanically strong double network
photo cross linked hydrogels from N, N-dimethylacrylamide and glycidyl
methacrylated hyaluronan, Biomaterials 29 (2008) 2153e2163.
[39] Y.H. Bae, S.W. Kim, Hydrogel delivery systems based on polymer blends, block
co-polymers or interpenetrating networks, Adv. Drug Deliv. Rev. 11 (1993)
109e135.
[40] K.S.V. Krishna Rao, B. Vijaya Kumar Naidu, M.C.S. Subha, M. Sairam,
T.M. Aminabhavi, Novel chitosan-based pH-sensitive interpenetrating
network microgels for the controlled release of cefadroxil, Carbohydr. Polym.
66 (2006) 333e344.
[41] S. Suri, C.E. Schmidt, Photopatterned collagen-hyaluronic acid inter-
penetrating polymer network hydrogels, Acta Biomater. 5 (2009) 2385e2397.
[42] P. Matricardi, I. Onorati, G. Masci, T. Coviello, F. Alhaique, Semi-IPN hydrogel
based on scleroglucan and alginate: drug delivery behaviour and mechanical
characterization, J. Drug Deliv. Sci. Technol. 17 (2007) 193e197.
[43] K.I. Draget, O. Smidsrød, G. Skjåk-Bræk, Alginates from algae, in: S. De Baets,
E.J. Vandamme, A. Steinbüchel (Eds.), Biopolymers, vol. 6, Wiley-VCH, Wein-
heim, Germany, 2002, pp. 215e244.
[44] B.H.A. Rehm, Alginates from bacteria, in: S. De Baets, E.J. Vandamme,
A. Steinbüchel (Eds.), Biopolymers, vol. 5, Wiley-VCH, Weinheim, Germany,
2002, pp. 179e212.
[45] P. Matricardi, C. Di Meo, T. Coviello, F. Alhaique, Recent advances and per-
spectives on coated alginate microspheres for modified drug delivery, Expert
Opin. Drug Deliv. 5 (2008) 417e425.
[46] Oddo, G. Masci, C. Di Meo, D. Capitani, L. Mannina, R. Lamanna, S. De Santis,
F. Alhaique, T. Coviello, P. Matricardi, Novel thermosensitive calcium alginate
microspheres physico-chemical characterization and delivery properties, Acta
Biomater. 6 (2010) 3657e3664.
[47] L. Pescosolido, T. Vermonden, J. Malda, R. Censi, W.J.A. Dhert, F. Alhaique,
W.E. Hennink, P. Matricardi, In situ forming IPN hydrogels of calcium alginate
and dextran-hema for biomedical applications, Acta Biomater. 7 (2011)
1627e1633.
[48] S.N. Pawar, K.J. Edgar, Alginate derivatization: a review of chemistry, prop-
erties and applications, Biomaterials 33 (2012) 3279e3305.
[49] K.Y. Lee, D.J. Mooney, Alginate: properties and biomedical applications, Prog.
Polym. Sci. 37 (2012) 106e126.
[50] P. Matricardi, M. Pontoriero, T. Coviello, M.A. Casadei, F. Alhaique, In situ
cross-linkable novel alginate-dextran methacrylate IPN, hydrogels for
biomedical applications: mechanical characterization and drug delivery
properties, Biomacromolecules 9 (2008) 2014e2020.
[51] L. Pescosolido, S. Miatto, C. Di Meo, C. Cencetti, T. Coviello, F. Alhaique,
P. Matricardi, Injectable and in situ gelling hydrogels for modified protein
release, Eur. Biophys. J. 39 (2010) 903e909.
[52] G. D'Arrigo, C. Di Meo, L. Pescosolido, T. Coviello, F. Alhaique, P. Matricardi,
Calcium alginate/dextran methacrylate IPN beads as protecting carriers for
protein delivery, J. Mater. Sci. Mater. Med. 23 (2012) 1715e1722.
[53] L. Pescosolido, T. Vermonden, J. Malda, R. Censi, W.J.A. Dhert, F. Alhaique,
W.E. Hennink, P. Matricardi, In situ forming IPN hydrogels of calcium alginate
and dextran-HEMA for biomedical applications, Acta Biomater. 7 (2011)
1627e1633.
[54] L. Pescosolido, T. Piro, T. Vermonden, T. Coviello, F. Alhaique, W.E. Hennink,
P. Matricardi, Biodegradable IPNs based on oxidized alginate and dextran-
HEMA for controlled release of proteins, Carbohydr. Polym. 86 (2011)
208e213.
[55] Y. Hu, T. Chen, X. Dong, Z. Mei, Preparation and characterization of composite
hydrogel beads based on sodium alginate, Polym. Bull. (30 June 2015) 13,
http://dx.doi.org/10.1007/s00289-015-1440-2 (in press).
[56] G. D'Arrigo, C. Di Meo, E. Geissler, T. Coviello, F. Alhaique, P. Matricardi, Hy-
aluronic acid methacrylate derivatives and calcium alginate interpenetrated
hydrogels networks for biomedical applications: physico-chemical charac-
terization and protein release, Colloid Polym. Sci. 290 (2012) 1575e1582.
[57] L. Pescosolido, L. Feruglio, R. Farra, S. Fiorentino, I. Colombo, T. Coviello,
P. Matricardi, W.E. Hennink, T. Vermonden, M. Grassi, Mesh size distribution
determination of interpenetrating polymer network hydrogels, Soft Matter 8
(2012) 7708e7715.
[58] E.E. Hago, L. Xinsong, J.J. Kai, A new route toward IPN photocrosslinked
alginate hydrogels with biodegradation rates, excellent biocompatibility and
mechanical properties, J. Biomater. Tissue Eng. 4 (2014) 89e98.
[59] F. Corrente, H.M. Abu Amara, S. Pacelli, P. Paolicelli, M.A. Casadei, Novel
injectable and in situ cross-linkable hydrogels of dextran methacrylate and
scleroglucan derivatives: preparation and characterization, Carbohydr. Polym.
92 (2013) 1033e1039.
[60] E. Amici, A.H. Clark, V. Normand, N.B. Johnoson, Interpenetrating network
formation in gellan-agarose gel composites, Biomacromolecules 1 (2000)
12 F. Alhaique et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e12
721e729.
[61] R.V. Kulkarni, B.S. Mangond, S. Mutalik, B. Sa, Interpenetrating polymer
network microcapsules of gellan gum and egg albumin entrapped with
diltiazem-resin complex for controlled release application, Carbohydr. Polym.
83 (2011) 1001e1007.
[62] S. Pacelli, P. Paolicelli, I. Dreesen, S. Kobayashi, A. Vitalone, M.A. Casadei,
Injectable and photocross-linkable gels based on gellan gum methacrylate: a
new tool for biomedical application, Int. J. Biol. Macromol. 2015 (72) (2015)
1335e1342.
[63] J.P. Gong, Why are double network hydrogels so tough? Soft Matter 6 (2010)
2583e2590.
[64] S. Pacelli, P. Paolicelli, F. Pepi, B. Tita, A. Vitalone, M.A. Casadei, Gellan gum and
polyethylene glycol dimethacrylate double network hydrogels with improved
mechanical properties, J. Polym. Res. 21 (2014) 1e13.
[65] A.V. Kabanov, S.V. Vinogradov, Nanogels as pharmaceutical carriers: finite
networks of infinite capabilities, Angew. Chem. Int. Ed. 48 (2009) 5418e5429.
[66] N. Zhang, P.R. Wardwell, R.A. Bader, Polysaccharide-based micelles for drug
delivery, Pharmaceutics 5 (2013) 329e352.
[67] K. Akiyoshi, S. Yamaguchi, J. Sunamoto, Self-aggregates of hydrophobic
polysaccharide derivatives, Chem. Lett. 20 (1991) 1263e1266.
[68] K. Akiyoshi, S. Deguchi, N. Moriguchi, S. Yamaguchi, J. Sunamoto, Self-aggre-
gates of hydrophobized polysaccharides in water. Formation and character-
istics of nanoparticles, Macromolecules 26 (1993) 3062e3068.
[69] K. Akiyoshi, J. Sunamoto, Supramolecular assembly of hydrophobized poly-
saccharides, Supramol. Sci. 3 (1e3) (MarcheSeptember 1996) 157e163.
[70] K. Akiyoshi, S. Kobayashi, S. Shichibe, D. Mix, M. Baudys, S.W. Kim,
J. Sunamoto, Self-assembled hydrogel nanoparticle of cholesterol-bearing
pullulan as a carrier of protein drugs: complexation and stabilization of in-
sulin, J. Contr. Rel 54 (1998) 313e320.
[71] K. Kuroda, K. Fujimoto, J. Sunamoto, K. Akiyoshi, Hierarchical self-assembly of
hydrophobically modified pullulan in water: gelation by networks of nano-
particles, Langmuir 18 (2002) 3780e3786.
[72] V.M. Platt, F.C. Szoka, Anticancer therapeutics: targeting macromolecules and
Please cite this article in press as: F. Alhaique, et al., From macro to nano polysaccharide hydrogels: An opportunity for the delivery of drugs,
Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.09.018
nanocarriers to hyaluronan or CD44, a hyaluronan receptor, Mol. Pharmacol. 5
(2008) 474.
[73] G. D'Arrigo, C. Di Meo, E. Gaucci, S. Chichiarelli, T. Coviello, D. Capitani,
F. Alhaique, P. Matricardi, Self-assembled gellan-based nanohydrogels as a
tool for prednisolone delivery, Soft Matter 8 (45) (2012) 11557e11564.
[74] E. Montanari, S. Capece, C. Di Meo, M. Meringolo, T. Coviello, E. Agostinelli,
P. Matricardi, Hyaluronic acid nanohydrogels as a useful tool for bsao
immobilization in the treatment of melanoma cancer cells, Macromol. Biosci.
13 (2013) 1185e1194.
[75] E. Montanari, G. D'Arrigo, C. Di Meo, A. Virga, T. Coviello, C. Passariello,
P. Matricardi, Chasing bacteria within the cells using levofloxacin-loaded
hyaluronic acid nanohydrogels, Eur. J. Pharm. Biopharm. 87 (2014) 518e523.
[76] C. Di Meo, E. Montanari, L. Manzi, C. Villani, T. Coviello, P. Matricardi, Highly
versatile nanohydrogel platform based on riboflavin-polysaccharide de-
rivatives useful in the development of intrinsically fluorescent and cyto-
compatible drug delivery systems, Carbohydr. Polym. 115 (2015) 502e509.
[77] G. D'Arrigo, C. Di Meo, E. Gaucci, S. Chichiarelli, T. Coviello, D. Capitani,
F. Alhaique, P. Matricardi, Self-assembled gellan-based nanohydrogels as a
tool for prednisolone delivery, Soft Matter 8 (2012) 11557e11564.
[78] E. Montanari, M.C. De Rugeriis, C. Di Meo, T. Coviello, F. Alhaique,
P. Matricardi, One-step formation and sterilization of gellan and hyaluronan
nanohydrogels using autoclave, J. Mat. Sci. Mater. Med. 26 (2015) 1e6.
[79] G. D'Arrigo, G. Navarro, C. Di Meo, P. Matricardi, V. Torchilin, Gellan Gum
nanohydrogel containing anti-inflammatory and anti-cancer drugs: a multi-
drug delivery system for a combination therapy in cancer treatment, Eur. J.
Pharm. Biopharm. 87 (2014) 208e216.
[80] E. Montanari, G. D'Arrigo, C. Di Meo, A. Virga, T. Coviello, C. Passariello,
P. Matricardi, Chasing bacteria within the cells using levofloxacin-loaded
hyaluronic acid nanohydrogels, Eur. J. Pharm. Biopharm. 87 (2014) 518e523.
[81] S. Sharmin, K. Sakata, K. Kashiwagi, S. Ueda, S. Iwasaki, A. Shirahata,
K. Igarashi, Polyamine cytotoxicity in the presence of bovine serum amine
oxidase, Biochem. Biophys. Res. Commun. 282 (2001) 228e235.