Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223

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Colloids and Surfaces B: Biointerfaces

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

Hyaluronan based materials with catanionic sugar-derived

surfactants as drug delivery systems
F. Roig a , M. Blanzatb, C. Solansc,d, J. Esquenac,d, M.J. García-Celmaa,d,∗
a
Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica, IN2UB, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain
b
Laboratoire des IMRCP, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9,
France
c
Institut de Química Avançada de Catalunya (IQAC), Consejo Superior de Investigaciones Científicas (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
d
CIBER-BBN (Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina), Jordi Girona 18-26, 08034 Barcelona, Spain

a rti c l e i nf o a b s t r a c t

Article history: In the present work novel drug delivery systems consisting in highly porous Hyaluro nan foams for the
Received 23 December 2016 administration of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, have been obtained. A
Received in revised form 17 January 2018 sugar-derived surfactant associated with ketoprofen was prepared and incorporated into the porous
Accepted 20 January 2018
hyaluronan materials. The association between a lactose derived surfactant, Lhyd 12, and ketoprofen
was obtained by acid-base reaction and its physicochemical properties were studied. Tensiometric and
Keywords:
dynamic light scattering (DLS) determinations showed the formation of catanionic surfactant aggregates,
Hyaluronan
Lhyd12/ketoprofen, in aqueous solution. Furthermore, the catanionic surfactants allowed greater solu -
Catanionic surfactant
Controlled release bilisation of ketoprofen. Hyaluronan porous materials were developed using butanediol diglycidyl ether
Crosslinked hydrogel as crosslinking agent. The profile release of Lhyd 12/ketoprofen from hyaluronan based materials shows
Solid foam differences as a function of the aggregation state of catanionic surfactant.
Highly concentrated emulsion © 2018 Elsevier B.V. All rights reserved.
Ketoprofen

1. Introduction
Soft matter drug delivery systems have recently received sub-
stantial attention, in particular from the nanomedicine field. In
fact, these carriers increase drug bioavailability and activity, while
decreasing its toxicity, because drug efficiency is often altered by
its non-controlled biodistribution. The design of the appropriate
drug delivery system is then important to optimize drug efficiency
[1]. In order to meet the needs, a wide range of drug delivery sys-
tems have already been developed. These systems include matrix
and vesicular carriers, with a large range of size and structures
[2]. The use of a biocompatible polymer matrix is an interesting
approach because it allows controlling the release of low molec-
ular weight drugs for various routes of administration such as:
oral, parenteral, ocular, etc. [3]. Furthermore, these materials have
the following advantages: reduction of side effects and improve-
ment of drug bioavailability, solubilization of lipophilic drugs, and
∗ Corresponding author at: Departament de Farmàcia i Tecnologia Farmacèutica
i Fisicoquímica, IN2UB, Universitat de Barcelona, Joan XXIII s/n, 08028, Barcelona,
Spain.
E-mail address: mjgarcia@ub.edu (M.J. García-Celma).
lower treatment costs [4]. Hyaluronan (HA), belongs to the fam-
ily of glycosaminoglycans and consists on N-acetyl-d-glucosamine
and D-glucuronic acid [5]. This polymer is an important compo-
nent of the extracellular matrix of connective tissue and is found in
various parts of the human body such as: skin, cartilage, vitreous
humour and intra-articular joint fluid [6]. It also plays an impor-
tant role in cartilage matrix stabilization, cell proliferation, control
of morphogenesis, cancer metastases, inflammation processes and
wound healing [7–12]. HA is degradable in vivo by enzymes such as
hyaluronidase present in human tissues [13]. A suitable approach
to avoid fast elimination from the human body could be the prepa-
ration of HA materials chemically crosslinked that show an increase
in their resistance against hyaluronidase [14,15].
Polymeric materials can be obtained by crosslinking hydrophilic
polymers in bulk [16] (hydrogels) or by the use of colloidal systems
as templates for the preparation of materials with controlled poros-
ity (solid foams). The incorporation of a polymer in the continuous
phase of a highly concentrated emulsion, allows the preparation
of porous materials with very high pore volume [17]. These mate-
rials have found a number of applications, including biomaterial
engineered devices and drug delivery systems [18].
In addition, vesicular drug delivery systems made of catanionic
surfactants have also proved their great contribution to drug solu-

https://doi.org/10.1016/j.colsurfb.2018.01.037
0927-7765/© 2018 Elsevier B.V. All rights reserved.
 F. Roig et al. / Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223
bility in water and drug delivery by means of spontaneous vesicles
formation [19–21]. These vesicles also showed an increase of the
therapeutic effect of the drug together with a sustained diffusion
through the skin [19,22]. The strategy adopted in this work was
to evaluate the impact of the combination of both drug delivery
systems by preparing a novel complex drug delivery system. These
advanced materials consist in the association between a drug, keto-
profen (KP), and a sugar-derived surfactant to form an ion-pair,
“catanionic” surfactant, which would later be incorporated into a
crosslinked polymer matrix based on HA. To ensure the formation
of a KP catanionic surfactant, the selected surfactant has to be capa-
ble of forming an ionic acid-base pair with the carboxylic acid of KP.
This feature means that the surfactant requires the presence of a
basic group that is able to react with the acid group of the drug.
In addition, the surfactant has to be biocompatible and biodegrad-
able without inducing toxicity to ensure biocompatibility of the ion
pair within the body. Furthermore, the physicochemical character-
istics of the surfactant should lead to drug solubilization. In this
context, sugar-derived surfactants represent a suitable choice for
this purpose. Apart from their biocompatibility and biodegradabil-
ity, these surfactants can be obtained from sugars that are natural
raw materials available in large quantities.
The aim of this work was to develop a novel drug delivery system
based on HA materials (hydrogels and solid foams) loaded with KP
catanionic surfactants and determine the differences in the release
behavior when the aggregates of catanionic surfactants are formed.
2. Experimental
2.1. Materials
Hyaluronic acid sodium salt from Streptococcus equi. of molecu-
lar weight around 2 million Daltons with 97% purity was obtained
from Sigma-Aldrich. The chemical crosslinker butanediol diglycidyl
ether (BDDE) with a molecular weight 202.25 g mol −1 with 95%
purity was obtained from Sigma-Aldrich. Nonionic surfactant Cre-
mophor RH455 (CRH 455) with an HLB between 14 and 16 was
obtained from BASF. Miglyol 812, medium chain triglycerides, was
obtained from Fagron. Ketoprofen (C16H14O3), (KP), non-steroidal
anti-inflammatory drug (NSAIDs) used as anionic precursor surfac-
tant, was from Fagron with 99.8% purity. Phosphate buffer solution
pH 7.4, (PBS) was prepared from: KH 2PO4 from Fagron Iberica
S.A.V, Na2HPO4 from Probus S.A, NaCl from Acofarma®, and Milli-
Q® deionized water. Cellulose tubular membrane was purchased
from Orange Scientific. Its properties are a 12.000-14.000 Da nomi-
nal MWCO, and 20 µm wall thickness. Mobile phase for HPLC (pH
3.0) was prepared from 45% of aqueous phase comprising: Citric
acid from Acofarma® with 99.5% purity, NaCl from Acofarma®, NaOH
from Acofarma®, and Milli-Q® deionized water; and 55% of organic
phase acetonitrile obtained from Carlo Erba Reagents, with 99.9%
purity.
2.2. Methods
2.2.1. Synthesis of KP catanionic surfactant
The catanionic sugar-derived surfactant was obtained by an
acid–base reaction between equimolar amounts (0.66 mmol) of
cationic and anionic precursor surfactants in 30 mL of water. N-
dodecylamino-1-deoxylactitol, designated as Lhyd12, was used as
cationic precursor surfactant. Lhyd12 was obtained as previously
described from a reductive amination of dodecylamine with lactose
[23,24]. An NSAID, KP was used as the anionic precursor surfactant.
The two components were added in ultrapure water and stirred for
24 h. The resulting homogeneous solution was lyophilized. After
lyophilization a white powder corresponding to the catanionic
219
Fig. 1. Molecular structure of KP catanionic surfactant as a result of ionic association
between Lhyd12 and ketoprofen.
sugar-derived surfactant was obtained with a quantitative yield
(Fig. 1).
2.2.2. Physicochemical characterization of KP catanionic
surfactant
2.2.2.1. Fourier transform−infrared spectroscopy (FT-IR). Infrared
spectra were obtained with a Perkin-Elmer IR FT 1760X. KBr discs
with a concentration of 0.5 w/w% were prepared of KP catanionic
surfactant.
2.2.2.2. Surface tension measurements. The values of surface ten-
sion as a function of catanionic surfactant concentration were
measured by the Wilhelmy plate method using a Kruss Ten-
siometer Easy Dyne at 25.0 ◦C±0.1 ◦C. The catanionic solutions
were prepared by dissolving weighted amounts of dry catanionic
associations in ultrapure water. Solutions were stirred at room tem-
perature during a few minutes.
2.2.2.3. Dynamic light scattering (DLS). Dynamic light scattering
was performed using a Malvern Zetasizer Nano-ZS, ZEN3600, with
a measuring range of 0.5 nm to 10 µm. The light source used
was a He-Ne laser with a wavelength of 633 nm. The temper-
ature was regulated at 25.0 ◦C with a Peltier with an accuracy
0.1 ◦C. The measuring angle was 173◦. Samples of aqueous solu-
of ±
tions of KP catanionic surfactant (3.5×10−3 M) were introduced
into cells (pathway, 10 mm). The deconvolution of the measured
intensity autocorrelation function of the samples was realized with
the multiple narrow modes program that uses a non-negatively
constrained least squares (NNLS) fitting algorithm to obtain the
distribution of diffusion coefficients (D) of the solutes. The appar-
ent equivalent hydrodynamic diameter (dh) was determined using
the Stokes-Einstein equation. Hydrodynamic diameter values were
obtained from three different runs.
2.2.3. Preparation of chemically crosslinked hydrogels
For the preparation of HA hydrogels, 50 mg of sodium
hyaluronate were introduced into test tubes of 12 × 75 mm, to
which 500 µL of crosslinking solution, consisting of BDDE (5% v/v)
in alkaline media (0.2 M NaOH), were added. Then, the HA and the
crosslinking solution were stirred with a vortex till a homogeneous
mixing. The resulting mixture was incubated at 25 ◦C for 24 h and
the HA crosslinked hydrogel was obtained. The epoxy groups of
BDDE react with the hydroxyls present in the HA polymer [25].
2.2.4. In vitro cell viability analysis
Cell viability in the presence free BDDE crosslinker, BDDE
crosslinked hyaluronan hydrogels and KP catanionic surfac-
tant was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay [26]. For
each assay, HeLa cells were seeded in Dulbecco’s modified Eagle’s
medium supplemented with FBS and antibiotics. Then, the culture
medium was replaced with samples at the required concentra-
tions. 100 µL of BDDE solution at the same concentration that in the
hydrogel and the corresponding dilutions 1:5 and 1:10 v/v, 100 µL
220 F. Roig et al. / Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223
Fig. 2. IR spectrum in KBr tablet method for: a) an equimolar mixture of Lhyd 12 and KP, and b) KP catanionic surfactant.
of KP catanionic surfactant (0.14, 0.7 and 1.18 mM) or 25 mg of
hydrogel and the corresponding dilutions 1:5 and 1:10 p/v were
incubated in this medium. After incubation, the medium was with-
drawn and cells were seeded with fresh medium and incubated
again. Then, MTT reagent solution was added and DMSO was used
to dissolve the formazan crystals. Absorbance was measured at a
h = 590 nm. The results were expressed as viability percentages.
2.2.5. Preparation of chemically crosslinked solid foam
For the preparation of HA solid foam, a O/W highly concen-
trated emulsion (HIPRE) was used as template. The components
of the HIPRE were: aqueous component, a castor oil derivative
as surfactant (CRH 455), and an oil derived from caprylic and
capric fatty acids (Miglyol 812) as oil component. The HIPRE with
HA (5% in water) in the continuous phase was formed with a
high-performance dispersing equipment (Ultaturrax). After HIPRE
formation, BDDE as crosslinker agent was introduced and kept in
25 ◦C for 24 h to crosslinking HA. After crosslinking reaction of HA in
the continuous phase of HIPRE, the surfactant and oil were removed
by solvent extraction with ethanol and water for 12 h. Finally the
HA solid foam was freeze-dryied.
2.2.6. Determination of the specific surface area of the solid foam
The specific surface area of the solid foam was determined by
Nitrogen sorption experiments. Nitrogen adsorption and desorp-
tion isotherms at 77 K of the solid foam were obtained by using an
AUTOSORBTM IQ instrument. According to the BET theory [27], the
specific surface area of the solid foam was determined fitting the
BET equation to the adsorption curve.
2.2.7. Incorporation of the Lhyd12/ketoprofen into the HA
materials
The method used to incorporate the KP catanionic surfactant
into the HA materials was the permeation method: the HA materi-
als (hydrogels and solid foams) were prepared and then immersed
in a volume of 2 mL solution of KP catanionic surfactant in water,
which was completely absorbed by HA materials.
2.2.8. Release studies of ketoprofen from HA materials
In vitro release studies were carried out in a dissolution tester.
The equipment used was an Elite 8TM dissolution tester from Han-
son Research Corporation (USA). It consists of 8 dissolution vessels
immersed in a thermostatic bath. Each dissolution vessel had a
capacity of 150 mL and a setup for semisolid formulations called
Ointment cellwas incorporated in each vessel. A cellulose mem-
brane was placed in each ointment cell to separate the hydrogel
from the receptor solution. The receptor solution consisted of
100 mL of PBS (pH 7.4). The temperature of the receptor solution
was 37 ◦C. The stirring speed of the paddles in each dissolution
vessel was 25 rpm.
The HA materials were placed in the ointment cell, as described
previously [28]. Then, 150 mL of receptor solution (PBS) were
placed into the glass vessel. In order to determine the amount of
drug released as a function of time, 0.5 mL of receptor solution has
been removed for analysis and the same amount of PBS solution
was replaced. The release study lasted 24 h. Three replicates of each
formulation were assayed.
2.2.9. Determination of ketoprofen concentration by HPLC
KP released was analyzed by HPLC. The chromatographic system
consisted on Shimadzu equipment with a column Kromasil® 100-
5C18 purchased from Akzo Nobel with dimensions of 250 × 4.6 mm
and pore size of 5 µm, and a UV detector set at 233 nm for ketopro-
fen determination.
Separation was carried out at room temperature using 55% ace-
tonitrile and 45% aqueous phase (pH 3.0), as a mobile phase, with
flow rate of 1 mL/min, and injection volume of 50 µL. The KP reten-
tion time was about 7 min.
 F. Roig et al. / Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223
Fig. 3. Aqueous solution surface tension as a function of the logarithm of the con-
centration of KP catanionic surfactant.
3. Results and discussion
3.1. Synthesis and characterization of KP catanionic surfactant
A catanionic sugar-derived surfactant based on the associa-
tion between lactose-derived surfactant, Lhyd12, and KP has been
obtained by acid-base reaction as described previously [19]. This
reaction consists on the proton transfer between KP and the basic
aminosugar. Therefore, the carboxylic acid of KP was transformed
to carboxylate. The formation of the catanionic surfactant was first
assessed by infrared spectroscopy comparing the spectra of the
initial reagents with that of the association. Fig. 2 shows the disap-
pearance of group (C O) and (OC OH) corresponding to carboxylic
acid, 1692 cm−1 and 1228 cm−1 respectively, and the appearance of
the group (COO-st as) at 1576 cm−1 and (COO-st sy) to 1393 cm−1
for the carboxylate.
To study the behavior of the KP catanionic surfactant in aque-
ous solution, the surface activity was determined by surface tension
measurements. The plot of the aqueous solution surface tension as
a function of the logarithm of the concentration of KP catanionic
surfactant (Fig. 3) allowed to obtain the critical aggregation con-
centration (CAC) which was around 1.5 mM. The size distribution
of the aggregates was verified by Dynamic Light Scattering (DLS)
at a concentration of 3.5 mM. The results showed the formation of
a large distribution of aggregate population with a diameter cen-
Fig. 4. SEM images corresponding to a) freeze-dried hydrogels, and b) solid foams based on HA.
221
tered at 130 nm with a polydispersity index of 0.2, as shown as
Supporting information. All these results are in good agreement
with the physicochemical features of vesicles already reported for
these catanionic associations [19].
3.2. Preparation of drug delivery systems based on HA materials
The HA materials (hydrogels and solid foams) were obtained
as described in the experimental section, and either free KP or KP
catanionic surfactant were added into the polymer matrix. Due to
poor solubility of free KP in water, its incorporation was performed
using alcoholic solution and phosphate buffered solution (pH 7.4)
into solid foams and hydrogels respectively. However, the increase
in the solubility for KP in the association of KP catanionic surfactant,
allows its incorporation by swelling the polymer matrix with water
(containing KP catanionic surfactant). SEM images of the two HA
based materials show differences in their structure. Freeze-dried
hydrogels exhibit a disordered layered structure (Fig. 4a), which is
commonly observed after lyophilization, and it can be attributed
to the growth of ice crystals during freezing [29]. However, high
porous solid foams were obtained by crosslinking in the continu-
ous phase of the HIPREs (Fig. 4b). The solid foams showed smaller,
more homogeneous and well interconnected pores. Their pore size
is between 1 and 5 µm, being the largest macropores around 30 µm,
and the smallest around 0.5 µm.
Also, the specific surface area of the solid foam was determined
by Nitrogen sorption experiments (Fig. 5), and the material show a
high specific surface area, around 20 m2/g. This porous texture is a
replica of the structure of the highly concentrated emulsion used
as template.
Cytotoxicity studies were performed in order to determine the
biocompatibility of the prepared materials. The MTT test [26] was
applied to study the influence of various concentrations of the
free crosslinker BDDE, BDDE crosslinked hyaluronan hydrogels and
catanionic surfactant on HeLa cells viability, as an indication of tox-
icity. HeLa cells are a versatile model cell line that is widely used
in nanomedicine tests. When the cells viability is higher than 80%,
it can be considered that the sample assayed induces low cytotox-
icity. The results showed the toxicity of the free crosslinker BDDE
(cell viability lower than 11%), in contrast to the catanionic sur-
factant (cell viability higher than 90%) and the BDDE crosslinked
Hyaluronan hydrogel (cell viability higher than 85%), an indication
of absence of free BDDE in the medium. As the catanionic surfactant
and the BDDE crosslinked HA hydrogels assayed induce low cyto-
toxicity in HeLa human cell line, they could be proposed as good
222 F. Roig et al. / Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223
Fig. 5. Nitrogen adsorption-desorption isotherms at 77 K of a HA based solid foam.
Fig. 6. KP release profiles as a function of HA materials: in solution, from HA hydro-
gels, and from HA solid foams.
candidates for implant developments. The details of cytotoxicity
studies performed are showed as Supporting information.
3.3. Release studies of KP catanionic surfactant incorporated in
HA materials
The main purpose was to study the influence of the material
structure based on HA (hydrogels and solid foams) and the aggre-
gation state of catanionic surfactant (<CAC or >CAC) in KP release.
Although drug release experiments are not an indication of in vivo
behavior, they constitute a good approach to compare different
formulations. For comparative purposes, a KP solution was also
studied as a reference, to probe that the active is not retained in
the cellulose membrane used.
Firstly, we studied the release properties of KP from hydro-
gels and solid foams based on HA. The results are shown in Fig. 4.
Whereas 80% of KP was released after 24 h from hydrogels, only
about 20% was reached from solid foams (Fig. 6). The release pro-
files obtained, show a strong influence of the HA materials structure
in KP diffusion. These differences may be due to the high capacity
of HA based hydrogels to swell. The swelling of HA based hydrogels
during the in vitro test helps the diffusion of KP to the receptor solu-
Fig. 7. KP release profiles from HA hydrogels: KP catanionic surfactant without
aggregation (<CAC), KP catanionic surfactant aggregates (>CAC), and free KP.
tion. However, for the HA based solid foams, the retention could be
caused by the smaller pores present in these materials. The release
of KP from HA solid foams is much lower than that obtained with
other solids foams (e.g. polystyrene solid foams, that reach 70% after
24 h) [18].
Also we studied the release profile of KP catanionic surfactant as
a function of their state of aggregation. The release of the KP catan-
ionic surfactant incorporated to hydrogels and solid foams based on
HA to PBS solution were performed with two concentrations: [KP
catanionic surfactant] >CAC: 5.9 mM, and [KP catanionic surfactant]
<CAC: 0.7 mM.
The release profiles of KP catanionic surfactant aggregates (>
CAC), and KP catanionic surfactant without aggregation (< CAC)
from hydrogels, took place with no lag-time but revealed slight
differences (Fig. 7). Both showed a fast initial release which slows
down progressively after several hours, but the maximum amount
released was around 100% for KP catanionic surfactant without
aggregation and 75% for KP catanionic surfactant aggregates, after
25 h. The release profiles of KP catanionic surfactant from hydro-
gels were compared to the release profile of hydrogel loaded with
free KP (Fig. 7) which was closer to 70%.
Similar trends were observed with KP catanionic surfactant from
HA solid foams (Fig. 8). There is a slower release when KP catanionic
surfactant is aggregated (around 20% after 24 h), while the release
is faster when it is not aggregated (around 50% after 24 h).
The difference observed in the release of KP catanionic surfac-
tant depending on their state of aggregation may be caused by
the influence of the polar head consisting of lactose present in KP
catanionic surfactant, which helps the diffusion of the ion pair KP
catanionic surfactant to the receptor solution. This effect cannot be
observed when KP cationic surfactant forms vesicles. KP is retained
in the HA matrix in a similar way that occurs with free KP.
4. Conclusions
Catanionic surfactants have been obtained by the association
between sugar-derived surfactants and NSAIDs, KP. The synthesis
of these systems by acid-base reaction is quick and easy to obtain.
The use of sugar-derived surfactants allows the preparation of bio-
compatible and biodegradable systems, and increases the solubility
of lipid-soluble drugs.
The characterization of KP catanionic surfactant assemblies by
DLS, showed that they form vesicles in aqueous solution above
 F. Roig et al. / Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223
Fig. 8. KP release profiles from HA solid foams: KP catanionic surfactant without
aggregation (<CAC), KP catanionic surfactant aggregates (>CAC), and free KP.
a critical aggregation concentration, the size of these aggregates
being suitable for the drug release from a polymeric matrix system.
Concerning the KP release from the hyaluronan materials, it
appears that the presence of the catanionic assembly improve the
drug release capacity of the material to PBS receptor solution. How-
ever, the release of the KP catanionic surfactant from HA materials
depends on the structure of HA materials and on the aggregation
state of the catanionic entity. The vesicles seem to be constrained
within the polymer matrix limiting then the KP delivery. These
results show that the KP release profile can be modulated through
a change on the porosity of the HA material or by the aggregation
state of the active principle.
Acknowledgements
The authors wish to acknowledge the sponsorship of the Span-
ish Ministry of Economy and Competitivity (CTQ2016-80645-R
and CTQ2014-52687-C-1-P) and Generalitat de Catalunya (Grant
2014SGR1655). The authors also acknowledge Dr. M. Monge and
M. Bover for technical support.
223
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.colsurfb.2018.01.
037.
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