Full Paper

Thermosensitive Macroporous Cryogels

Functionalized With Bioactive Chitosan/
Bemiparin Nanoparticles

Hazel Peniche, Felisa Reyes-Ortega,* Mar
ıa R. Aguilar, Gema Rodr
ıguez,

Cristina Abradelo, Luis Garc
ıa-Ferna

ndez, Carlos Peniche, Julio San Roma
n

Thermosensitive macroporous scaffolds of poly(N-isopropylacrylamide) (polyNIPA) loaded

with chitosan/bemiparin nanoparticles are prepared by the free radical polymerization
in cryogenic conditions. Chitosan/bemiparin nanoparticles of 102
6.5 nm diameter are
prepared by complex coacervation and loaded into polyNIPA cryogels. SEM image reveal the
highly porous structure of cryogels and the integration of nanoparticles into the macroporous
system. Volume phase transition temperature (VPT) and total freezing water content of
cryogels are established by differential scanning calorimetry, and their porosity is determined
by image-NMR. Swelling of cryogels (above and below the VPT) is highly dependent on
nanoparticles concentration. In vitro release profile of bemiparin from cryogel is highly
modulated by the presence of chitosan. Bemiparin released from nanoparticles preserves its
biological activity, as shown
by the BaF32 cell proliferation
assay. Cryogels are not cyto-
toxic for the human fibroblast
cells and present excellent
properties for application on
tissue engineering and con-
trolled release of heparin.

1. Introduction
H. Peniche, Dr. C. Peniche Cryogels are polymeric macroporous materials obtained by
Centro de Biomateriales, Universidad de La Habana, 10400, cryotropic gelation of monomers or polymer precursors in
Havana, Cuba moderately frozen state. In the freezing process the reactive
Dr. F. Reyes-Ortega, Dr. M. R. Aguilar, Dr. G. Rodr
ıguez,
components initially soluble in the medium at room

ndez, Prof. J. S. Roma
Dr. L. Garc
ıa-Ferna
n
temperature, are concentrated in the remaining liquid
Instituto de Ciencia y Tecnolog
ıa de Pol
ımeros, CSIC, C/ Juan de la
Cierva, 3, 28006, Madrid, Spain microphase, where the polymerization reaction is produced
C. Abradelo at a very fast rate giving rise to a very strong porous matrix.
Departamento de Qu
ımica, Facultad de Farmacia, Universidad The crystals of frozen solvent (normally water) act as
CEU San Pablo, Urbanizacio
n Montepr
ıncipe, 28668 Boadilla del porogen during gel formation giving rise to a three-
Monte, Madrid, Spain dimensional network of interconnected macroporous after

ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com Macromol. Biosci. 2013, DOI: 10.1002/mabi.201300184 1
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defrosting.[1] Interconnected macroporous (or superma-
croporous), allow unrestricted diffusion of solutes of
practically any size, as well as mass transport of nano-
and even microparticles, therefore cryogels are attractive
matrices for biomedical applications and biotechnological
applications.[2,3] In this sense, Kumar et al.[4] isolated
urokinase using supermacroporous cryogel matrix. Bo €lgen
[5]
et al. evaluated tissue response of biodegradable macro-
porous cryogels of dextran modified with oligo L-lactide
bearing hydroxyethylmethacrylate (HEMA), with highly
open and interconnected pore structures. Kathuria et al.[6]
synthesized chitosan–gelatine cryogels for tissue engineer-
ing, Hwang et al.[7] obtained poly (ethylene glycol) cryogels
as cell scaffolds for cartilage repairing.
The formation of active macromolecular systems with
morphology of cryogels can be implemented by the
presence of ‘‘smart’’ or sensitive polymeric components,
in such a way that an additional effect on the dimensions of
the porous and the properties of the support walls can be
obtained. In this sense we consider that poly(N-isopropy-
lacrylamide) (polyNIPA) could contribute efficiently to the
preparation of robust porous matrices with modulated
sensitivity to the temperature with a volume phase
transition (VPT) at a critical temperature (Tc) about 34 8C
in aqueous media. Below Tc, polyNIPA hydrogels are
swollen, hydrated, and hydrophilic. Above Tc, the gels
shrink due to the distortion of the hydrophilic/hydrophobic
balance in the network structure.[8] Thermal sensitive
polyNIPA gels have attracted much attention for their
usefulness in designing thermosensitive drug delivery
systems, developing separation techniques in biotechnolo-
gy, agricultural processing, and sensors. These applications
require a fast response of the hydrogel to temperature. To
increase the response speed of the polyNIPA gels, various
techniques have been proposed, being one of the most
interesting the preparation of macroporous systems such as
the so-called cryogels.[9]
The control over the regenerative potential of tissue
engineering scaffolds has dramatically improved in recent
years, mainly by using drug releasing scaffolds or by
incorporating drug delivery devices into tissue engineering
scaffolds,[10] to provide not only the suitable mechanical
and structural support, but also have to actively guide and
control cell attachment, migration, proliferation, and
differentiation. This may be achieved if the functions of
scaffold are extended to supply biological signals able to
guide and direct cell function through a combination of
matricellular cue exposition and growth factors (GF)
sequestration and delivery.[11,12]
Heparin is a glycosaminoglycan that inhibits blood
coagulation. It is a potent antithrombin activator known to
bind many of the angiogenic GF including vascular
endothelial growth factor (VEGF) and fibroblast growth
factor 2 (FGF-2) through specific binding domains. It is also
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important in boosting cell signaling for new blood vessel
formation. Heparin has either been covalently linked to
polymers such as alginate and collagen or physically
trapped within chitosan in order to promote angiogenesis
in vivo by means of the sustained release of GF.
Rajangam[13] report the use of heparin to nucleate nano-
structures that can display these macromolecules on their
surfaces in order to more effectively bind and activate
angiogenic GF for cell signaling.
Bemiparin is a low molecular weight heparin (LMWH)
derived from unfractionated heparin (UFH) by chemical or
controlled enzymatic depolymerization. LMWH presents
important advantages if compared to UFH: enhanced
bioavailability after subcutaneous administration, pro-
longed half-life and more interesting pharmacokinetic
properties, reduced thrombocytopenia, and a more predict-
able dose response.[14]
Bemiparin has a molecular weight of 5500 Da, a half-life
of 5.3 h and an anti-FXa/anti-FIIa activity ratio (8:1) which
reduce the risk of bleeding and improve the antithrombotic
activity if compared with UFH. Currently, Bemiparin is
licensed for the treatment and prophylaxis of venous
thromboembolism, and is the only LMWH licensed in
Europe for starting thromboprophylaxis after either
general or orthopedic surgery. Therefore, its use is safer
than UFH.
Moreover, several authors have demonstrated an impor-
tant role in the modulation of the activity of proangiogenic
GF such as FGF and VEGF.[15] Therefore, its use in this work is
fully justified in spite of its lower molecular weight and
weaker molecular interaction with chitosan.
Proteins such as fibronectin, gelatin, and small oligopep-
tides mimicking large proteins can be chemically or
physically modified to provide specific biological sites for
the immobilization of GF or morphogens. Small molecules
mimicking key fragments and functions of these large
proteins can also be used in their place to immobilize
GF.[16,17] The association of heparin with GF and specifically
the VEGF, has been demonstrated and important effect of
angiogenesis has been attributed to this specific associa-
tion.[18–20] Therefore the addition of both bioactive
ingredients in the same support systems could give
interesting effects on the bioactivity of the associated
polymer components.
The complexation between chitosan and oppositely
charged macromolecules leads to the formation of poly-
electrolyte complexes that can be used for designing
biomedical devices for drug delivery and bioactive agent’s
administration. These polyelectrolyte complexes can be
applied as films, sponges, powders, nano- and micro-
particles, among other forms.[21,22] Reyes-Ortega et al.[23]
described the preparation of methacrylic block copolymers
as bemiparin carriers to improve the LMWH stability and
modulate its biological activity. Nanoparticles based on
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Thermosensitive Macroporous Cryogels Functionalized with Bioactive Nanoparticles
chitosan and bemiparin polyelectrolyte complexation have
been prepared in this work. The incorporation of these
nanoparticles into macroporous scaffolds may be beneficial
in tissue engineering applications and regenerative medi-
cine, due to the ability of bemiparin loaded nanoparticles to
promote locally the activation of heparin-dependent GF
(VEGF or FGF).[24,25] In addition, the incorporation of
chitosan to the cryogel matrix brings a better compatibility
with the nanoparticles, and potential resorbability of the
polymeric support.
The aim of this work was the preparation and
characterization of thermosensitive macroporous scaffolds
obtained by cryopolymerization of N-isopropylacrylamide
(NIPA), and loading of porous hydrophilic scaffolds with
chitosan–bemiparin nanoparticles to incorporate their
valuable properties for tissue engineering applications in
a targeted and controlled way.
The application of cryogels can be considered a very
advanced and well-designed methodology, which controls
the porosity and connectivity of porous in hydrophilic
polymeric matrices prepared in very mild conditions
(a polymerization temperature of –12 8C). This is an
important factor because we have observed that the
characteristics of heparin change when the system is
treated at temperatures higher than 40 8C. In this work, it
has been applied the experimental conditions to obtain a
porous polymer system with adequate porous size and
interconnectivity.
2. Experimental Section
2.1. Materials
Chitosan M ¼ 150 000–400 000 g  mol1, 83% DD was purchased
from Novamatrix. Bemiparin (sodium salt, 111.3 UI.mg1, 5500 D)
was purchased from ROVI. NIPA, N,N-methylenebisacrylamide
(MBA), ammonium persulfate (APS) and N,N,N0 ,N0 -tetramethyle-
nediamine (TEMED), were all purchased from Aldrich Chemical Co.
NIPA was recrystallized from ether/hexane (1:5 v/v). Thermanox
(TMX) control discs were supplied by Labclinics S. L. and plasticware
by Sarstedt. Tissue culture media, additives, trypsin, phosphate
buffered saline (PBS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) were supplied by Sigma. Alamar
Blue (AB) was supplied by SEROTEC. PBS buffer (140 mM NaCl, 10 mM
phosphate buffer, 3 mM KCl), and buffer pH 10 (boric acid/
potassium chloride/sodium hydroxide) were purchased from
Merck-Millipore in powder and they were solved in 1 and 0.5 L
of distilled water, respectively. Recombinant human FGF-2
(Invitrogen) was used without further purifications. Dye CellTiter
96 Aqueous One Solution Reagent ([3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], in-
ner salt, MTS) (Promega) was also used as received. BaF32 cells are
an IL-3-dependent and HP sulfate proteoglycan deficient myeloid B
cell line that has been stably transfected with FGF receptor 1c
(FGFR-1c). BaF32 cells represent a model system developed to
identify HP/Heparan sulfate structures that interact with FGFs and
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their receptors to form active ternary complexes. RPMI 1640
powder culture medium (Sigma–Aldrich) was supplemented with
2 g  L1 sodium bicarbonate (Sigma–Aldrich, 99–100% purity,
suitable for cell culture), 10% (v/v) fetal bovine serum (FBS) (Gibco),
a 1% (v/v) penicillin/streptomycin mixture (Gibco), and the
conditioned medium was stored at 20 8C until it was required.
All other chemicals reagents and solvents were analytical grade.
2.2. Methods
2.2.1. Preparation of Chitosan/Bemiparin Nanoparticles
The nanoparticles were prepared by a complex coacervation
method at room temperature. In brief, 1.5 mL of a 0.1% (w/v)
chitosan solution in 1% (w/v) acetic acid (pH adjusted to 5) were
added to an aqueous bemiparin solution [0.03% (w/v), 5 mL, pH 5]
with constant stirring using an ultra-turrax homogeneizer
(IKA T25) at 3400 rpm. The resulting dispersion was dialyzed
against water (pH 5.5–5.8) for 3 d changing the water every 12 h.
The nanoparticles were isolated by freeze drying.
2.2.2. Characterization of Nanoparticles and Cryogels
The particle size distribution was determined by dynamic light
scattering (DLS) with a Zetasizer NanoZS (Malvern Instruments, UK)
equipped with a He–Ne laser beam with wavelength of 633 nm and
a scattering angle of 1738. The nanoparticle suspension (0.01 mg
mL1) were prepared in 25 mM HEPES buffer. For each sample, the
statistical average and standard deviation (SD) of data were
calculated from at least three measurements.
Scanning electron microscopy (SEM) was used to establish the
morphology of the freeze-dried nanoparticles and cryogels
obtained. SEM analysis was performed with a Philips XL30 field
emission scanning electron microscope. Nanoparticles were also
analyzed by atomic force microscopy (AFM). AFM was performed in
tapping mode using a Multimode AFM (Veeco Instruments, Santa
Barbara, CA, USA) with a Nanoscope IVa control system (software
version 6.14r1), equipped with silicon tapping probes (RTESP,
Veeco) having a resonance frequency 300 KHz and a scan rate of
0.5 Hz. Fourier transform infrared attenuated total reflectance
(FTIR-ATR) spectroscopy was performed in a Perkin Elmer Spectrum
One FTIR spectrometer. Transmission FTIR was applied to
determine the average content of bemiparin in the nanoparticles
using a normalize calibration protocol, by 32 scans and with a
resolution of 4 cm1. To estimate the compositional ratio of
complexed bemiparin and the average content in the nano-
particles, the absorption bands ratio A1521/A1220 were applied
according to the literature.[26]. The band at 1521 cm1 is related to
the amino group of chitosan and the band at 1220 cm1 is related to
the sulfate group of bemiparin. A previous calibration curve was
done using physical mixing of chitosan and bemiparin at different
ratios per triplicate. The FTIR spectra were obtained by KBr slabs
prepared of each mixture.
2.2.3. In Vitro Release Study
For the in vitro release experiments 5 mg of nanoparticles
were added into a vial with 3 mL of the corresponding buffer
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(PBS pH 7.4 or buffer pH 10). The particles were resuspended with an
ultrasound probe (1 min, 30 A). The vials were placed in a constant
temperature oven at 37 8C shaking at 300 rpm (digital IKA MTS 2/4
shaker). Aliquots (0.5 mL) were taken at different times and
replaced with the same volume of PBS solution. The aliquots were
centrifuged at 1200 rpm for 20 min and the supernatant was
separated for analysis. The pellet was resuspended with 0.5 mL of
fresh PBS solution and placed into the original nanoparticle
solution. Supernatants were analyzed by high performance liquid
chromatography (HPLC) in a SHIMADZU SIL-20 apparatus at
242 nm, using H2O at pH 3 [adjusted with 1% (w/v) acetic acid
solution] as mobile phase to determine the amount of bemiparin
released. Each experiment was performed with three replicates. A
calibration curve for bemiparin was previously obtained from
solutions of known concentration in the same medium, integrating
the peak at 2.3
0.15 min.
2.2.4. Baf32 Cell Proliferation Assay
The Baf32 cell proliferation assay was used to determine the
activity of the nanoparticle systems. The readout of this assay was
cell proliferation which indicated the biological activity of free and
complexed bemiparin by the formation of ternary complexes on
the cell surface where molecular interactions (mainly ionic) and
geometry participate.[27,28] Baf32 cells were maintained in RPMI
1640 medium containing 10% (v/v) FBS, 10% (v/v) WEHI-3BD
conditioned medium, and a 1% (v/v) penicillin/streptomycin
mixture. WEHI-3BD cells were maintained in RPMI 1640 medium
supplemented with 2 g L1 sodium bicarbonate, 10% (v/v) FBS, and
a 1% (v/v) penicillin/streptomycin mixture, and the conditioned
medium was collected three times per week and stored at –20 8C
until it was required. For the mitogenic assays, the Baf32 cells were
transferred into IL-3 depleted medium for 24 h prior to experimen-
tation and seeded into 96-well plates at a density of 8  104 cells per
well in the presence of free bemiparin or encapsulated bemiparin at
different concentrations and with and without FGF2 (0.03 nM). Cells
in the presence of medium without any treatment were used as a
negative control. Cells were incubated for 72 h in 5% CO2 at 37 8C,
and the number of viable cells was assessed using the MTS assay.
The MTS reagent was added to the cell cultures 6 h prior to
measurement of the absorbance at 490 nm. Cell proliferation was
assayed by measuring the increase of absorbance, which corre-
sponded to the cell number. To compare the difference between free
and complexed bemiparin, analysis of variance (ANOVA) of the
results were carried out using p < 0.05 of significance level.
2.2.5. Synthesis of Cryogels
Cryogels were prepared by the free radical cryopolymerization of
the NIPA in water and with small amount of bisacrylamide (BAM).
Polymerization reactions were carried out in tubular-shape
polypropylene molds. NIPA (1.2 g) was dissolved in water
(11 mL). A 2% w/v chitosan solution (11 mL,) in 1% w/v acetic acid
containing chitosan/bemiparin nanoparticles in different propor-
tions [0,1, 2, and 5% (w/v)] was added. The crosslinker, BAM (3.3 wt
% with respect to the monomer) was added to the solution and
nitrogen was bubbled through the solution for 15 min. TEMED
(2.7 wt% with respect to the monomer) was added and the solution
was cooled in an ice bath for 5 min. APS (2.6  103 wt% with
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respect to the monomer), was added and the reaction mixture was
stirred during 1 min. 1 mL of the reaction mixture was injected into
the mold. The solution was frozen at 20 8C for 1 h and then at
12 8C during 16 h. The obtained cryogels were freeze-dried. They
are designated as polyNIPA-CHI, and the nanoparticles content is
indicated in brackets.
Cryogels without chitosan (polyNIPA) were prepared as above,
but replacing the nanoparticle containing chitosan dispersion by a
dispersion of the NP in water. The cryogels obtained were cut into
discs of 5 mm diameter and 3 mm height. The discs were loaded
with nanoparticles by soaking them with 5 and 15 wt% nano-
particles dispersions in water with a micropipette.
2.2.6. Swelling Studies
Dried cryogel discs were weighed accurately. These fragments were
placed into flasks with 10 mL of PBS buffer solution (pH 7.4) and
kept in a thermostated bath subjected to successive cycles of 2 h at
15 8C (bellow the VPT) and 2 h at 37 8C (above the VPT). The
hydration percentage, %H, was calculated by measuring the weight
of the samples at different times after carefully wiping the surface
with a filter paper. It was reported as
Wt  Wo
%HðhydrationÞ ¼  100 ð1Þ
Wo
where Wt is the cryogel weight at time t and Wo is the weight
of the dry cryogel.
2.2.7. DSC
The VPT temperature of the cryogels was determined with a
PerkinElmer DSC-7 calorimeter. Measurements and calibration
were carried out at a heating rate of 10 8C min1. VPT temperature
was taken as the onset point of the transition region. Samples (13–
36 mg) were introduced into aluminum pans and heated from 20 to
50 8C, before the experiments were performed.
2.2.8. iNMR
Cylindrical samples of 5 mm diameter and 3 mm height were
immersed in vials containing PBS solution. The vial was
depressurized several times inside a desiccator in order to eliminate
air bubbles and was maintained under vacuum until used. NMR
images were processed using NIH Image (NIH, Bethesda, MD) and
Bruker Paravision software. Hahn spin echo images were acquired
at echo times (tE) of 4.349, 10, 20, 40, 80, and 120 ms at temperatures
of 21, 24, 28, 32, 34, and 36.5 8C. Several representative regions of
interest (ROI) were defined avoiding non-homogeneous areas in B1
(artifacts) and the mean signal intensity measured for each sample,
medium, and background. The average proton density for each
sample and the medium was determined by fitting the NMR data to
a mono-exponential function and extrapolating to tE ¼ 0. The
apparent porosity was calculated according to the equation:
Pi ð%Þ ¼ ½ðSi  Sb Þ=ðSm  Sb Þ  100 ð2Þ
where Pi represents the apparent open porosity (i.e., open volume
within the sample connected to the outside), Ssi, Sb, and Sm are the
mean signal intensities of a sample section, background, and
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Thermosensitive Macroporous Cryogels Functionalized with Bioactive Nanoparticles
medium, respectively. All experiments were carried out at least in
triplicate.[29]
2.2.9. Biocompatibility Assay
TMX and NIPA-CHT cryogels (cylindrical samples of 5 mm diameter
and 3 mm height) with nanoparticles content of 0, 1, 2, and 5%, were
set up in 5 mL of DMEM. They were placed on a roller mixer at 37 8C
and the medium was removed at different time periods (1 h, 2 h, 1 d,
2 d, 7 d, and 15 d) and replaced with other 5 mL of fresh medium. All
the extracts were obtained under sterile conditions. 100 mL of
fibroblasts cells were seeded at a density of 8  104 cells mL1 in
complete medium and placed in a sterile 96-well culture plate and
incubated. Then, the medium was replaced with the corresponding
eluted extract and incubated at 37 8C for 24 h. A solution of MTT was
prepared in warm PBS (0.5 mg  mL1) and the plates were
incubated at 37 8C for 4 h. Excess medium and MTT were removed
and 100 mL of dimethylsulfoxide (DMSO) were added to all wells in
order to dissolve the MTT taken up by the cells. This was mixed for
10 min and the absorbance was measured with a Biotek Synergy
HT detector using a test wavelength of 570 nm and a reference
wavelength of 630 nm.
3. Results and Discussion
3.1. Characterization of Chitosan/Bemiparin
Nanoparticles
Nanoparticles were produced by the spontaneous inter-
polyelectrolyte complexation of polycationic chitosan and
polyanionic bemiparin. The FTIR spectrum of nanoparticles
(not shown) exhibited the characteristic absorption bands
of chitosan at 1658 cm1 (Amide I), 1595 cm1 (—NH2
bending), and 1314 cm1 (Amide III). The presence of
bemiparin was evidenced by a band at 1240 cm1
corresponding to the antisymmetric stretching vibrations
nas (S—O) of the sulfate group.
Dynamic light scattering determination revealed that
under the experimental conditions of the present work a
Figure 1. Size distribution of chitosan/bemiparin polyelectrolyte complex particles obtained by DLS under the present experimental
conditions.
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monomodal particle size distribution was obtained with an
average size of 102
6.5 nm and 99.9% of particles less
than 1 mm (Figure 1).
The morphology of particles, analyzed by SEM and
AFM (Figure 2A,B) revealed that the particles in the dry
state have homogeneous size distribution and almost
spherical shape.
3.2. Release of Bemiparin From Chitosan/Bemiparin
Nanoparticles
The release profiles of bemiparin from chitosan/bemiparin
nanoparticles at 37 8C in PBS at pH 7.4 and 10 are shown in
Figure 3. The cumulative bemiparin release is normalized to
its total content in the nanoparticles.
At pH 7.4 only 0.85 mg of the bemiparin was released
during the first 72 h, however at pH 10 the release reached
2.74 mg. This is due to a pH-dependent complexation of the
polyelectrolyte (Scheme 1) because of the weak polybasic
nature of chitosan.
At pH 7.4 the pKa of chitosan can be considered close to
the intrinsic pK, pKa ¼ 6.5,[30] and use can be made of the
classical equation:
a
pK a ¼ pH þ log ¼ 6:5 ð3Þ
1a
where a is the degree of dissociation of chitosan, which
at pH 7.4 is about 14%.[31] These cationic charges on the
chitosan chain are sufficient to support the polyelectrolyte
complex formed with bemiparin, whose sulfate groups can
be considered as fully dissociated whatever the pH.
However, at pH 10 almost all amino groups of chitosan
are uncharged, and the complex breaks down releasing
bemiparin. These results indicate the relatively high
stability of the polyelectrolyte complex in physiological
conditions and, not only ionic interactions but also weak
interactions (e.g., hydrogen bonds and macromolecular
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Figure 2. Morphology of chitosan/bemiparin nanoparticles: A) SEM image; B) AFM image.

3.5 Free Bemiparin

Free Bemiparin + FGF2
3 Bemiparin-Chitosan NP
2,6
Bemiparin-Chitosan NP + FGF2
2,4
2.5 2,2
mg bemiparin released

2,0
2 1,8
Absorbance (AU)

1,6

1.5 pH 7.4
1,4

pH 10 1,2

1 1,0

0,8

0.5 0,6

0,4

0 0,2
0 10 20 30 40 50 60 70 80
0,0
me (h) 5 10 30 60 5 10 30 60
Free Bemiparin (nM) Bemparin (nM) in Chitosan NP
Figure 3. Release behavior of chitosan/bemiparin nanoparticles
in buffered at 37 8C: pH 7.4 (*); pH 10 (&). Figure 4. Proliferation of Baf32 cells expressing FGFR 1c in
the presence of free bemiparin and bemiparin–chitosan
nanoparticles, either in the presence or absence of FGF2.
OH - Nanoparticle system was analyzed at a range of
NH +3 ….. - O3 SOCH2 NH 2 + - O3 SOCH2 + H2O concentrations based on the amount of complexed bemiparin
at the start of the assay. Data presented as mean
SD and
corrected for the proliferation of cells in the presence of
Scheme 1. Effect of pH on the stability of the chitosan/bemiparin medium only (n ¼ 3). Statistical analysis was performed using a
polyelectrolyte complex. one-way ANOVA, using a p < 0.05.

entanglements) are responsible of the high nanoparticle
stability.
3.3. Cell Proliferation Assay
The Baf32 proliferation assay provides a measure of the
activity of the ternary complexes formed between bemi-
parin, FGF2, and FGFR-1c. The formation of this active
ternary complex induces cell proliferation which was
measured by the MTS assay. The extent of proliferation is
modulated by the concentration of FGF2 and bemiparin as
well as the activity of the bemiparin. FGF2 or bemiparin
alone can induce the proliferation of the cells; however the
presence of both in an active ternary complex enhances
proliferation. This was observed when the Baf32 cells
were exposed to free bemiparin in the concentration range
of 5–60 nM in the presence or absence of FGF2 (Figure 4).
There was no dose-dependent increase in the proliferation
of the cells in the presence of increasing concentrations of
bemiparin in the range of 5–60 nM. Bemiparin–chitosan
nanoparticles were also analyzed using the Baf32
proliferation assay where cells were exposed to different

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Thermosensitive Macroporous Cryogels Functionalized with Bioactive Nanoparticles
concentrations of the nanoparticles based on the amount of
complexed bemiparin in the same range of concentrations
as described for the free bemiparin. The results obtained
when bemiparin–chitosan nanoparticles were added to the
culture medium showed no statistical differences (p < 0.05)
to the results obtained with free bemiparin. Therefore,
these results demonstrate that bemiparin keeps its
biological activity to form the ternary complex bemi-
parin–FGF2–FGF1c when it is ionically complexed to
chitosan in the range of the studied concentrations.
However, similar studies carried out by our group
demonstrated that encapsulated bemiparin nanoparticles
with none or less ionic complex interactions between
bemiparin and the corresponding polymer showed a dose-
dependent behavior with bemiparin concentration.[23] So,
we have described different polymer-bemiparin nano-
particles with not only modulated but also similar
biological activity than free bemiparin. The main advan-
tage of bemiparin base nanoparticles is that the active
nanoparticles can be included into a polymeric matrix (e.g.,
PNIPA cryogels) to be used for several biotechnological
or biomedical applications, as tissue engineering
scaffolds or the release of heparin dependent GF (e.g., FGF
and VEGF).
3.4. Characterization of Cryogels
3.4.1. FTIR-ATR Analysis
FTIR-ATR spectrum of the polyNIPA cryogel loaded with the
chitosan/bemiparin nanoparticles is shown in Figure 5,
together with the corresponding spectrum of the non-
Transmitance
1240
1060
2000 1800 1600 1400 1200
-1
ν (cm )
Figure 5. FTIR-ATR spectra of polyNIPA cryogels: unloaded (broken
line) and loaded (solid line) with chitosan/bemiparin
nanoparticles. The characteristic stretching vibration bands of
heparin are indicated therein.
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loaded polyNIPA cryogel (broken line). The presence of
nanoparticles in the loaded cryogel is manifested by the
presence of the band at 1240 cm1 corresponding to
the antisymmetric stretching vibrations nas (S—O) of the
sulfate group of bemiparin and the presence of the
absorption bands due to symmetric vibrations of sulfate
and antisymetric and symmetric vibrations of C—O—S
groups located at 1060, 1000, and 810 cm1, respectively.[32]
3.4.2. Swelling Kinetics
The thermoreversible behavior of polyNIPA gels is man-
ifested by a VPT at a temperature close to 34 8C, going from a
swollen state below VPT to a contracted one above VPT. In
order to investigate the effect of chitosan/bemiparin
nanoparticles on the swelling of polyNIPA cryogels,
samples with different nanoparticles content (expressed
in % w/v in Figure 6) were subjected to two successive
swelling–shrinking cycles. In Figure 6A cryogels at 15 8C
(below the VPT) are highly swollen and exhibit the expected
contraction when placed at 37 8C (above the VPT). The
reversibility of the process is verified in the second
temperature cycle. However, no influence of the nano-
particles content is manifested in the swelling behavior of
the cryogels.
The addition of a 1% chitosan solution in the preparation
of polyNIPA cryogels modifies the swelling degree at 15 8C
(Figure 6B) due to specific interactions of the hydrophilic
and polar groups of the chitosan and the P(NIPA) chains
formed by the polymerization in the medium to give a
strong semi-interpenetrating polymer network. The addi-
tion of 1–5 wt% nanoparticles to this polymer matrix,
modifies noticeably the swelling degree. This is clearer at
15 8C (below the VPT) than at 37 8C (above the VPT) and
seems to be repetitive after several cycles of variation of the
temperature.
3.4.3. Morphology of polyNIPA Cryogels
The image of the polyNIPA cryogel in Figure 7A reveals the
highly porous structure of the samples obtained by the
cryopolymerization procedure with a homogeneous distri-
bution and connectivity of pores. The cryogel loaded with
810
chitosan/bemiparin nanoparticles shown in Figure 7B
reveals that in the absence of chitosan the nanoparticles
are deposited on the surface of the gel, while in the presence
of chitosan they are more integrated into the pore gel walls
1000
(Figure 7C). This explains why in the former case the
particles exert almost no influence whatsoever on the
1000 800 600 swelling behavior of the cryogels. In the presence of
chitosan, the integration of the nanoparticles to the gel
reinforces its structure, increasing its rigidity with the
consequent decrease in maximum swelling (Figure 5B). This
effect is enhanced at higher nanoparticle concentrations as
already observed.
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Figure 6. Influence of the content of chitosan/bemiparin nanoparticles (NP) on the swelling-deswelling behavior of polyNIPA cryogels in
PBS (pH 7.4): a) cryogels prepared without chitosan (polyNIPA) NP content: (&) 0%; (*) 5%; (~) 15%; b) cryogels prepared with the addition
of 1% chitosan (polyNIPA-CHI). NP content: (&) 0%; (~) 1%; (*) 2%; (*) 5%.

Figure 7. SEM images of polyNIPA cryogels: A) unloaded; B) loaded with (5%) chitosan/bemiparin nanoparticles without chitosan [polyNIPA
(5)]; and C) with chitosan [polyNIPA-CHI(5)].

3.4.4. Volume Phase Transition Temperature
Determination
Volume phase transition temperatures were determined by
differential scanning calorimetry (DSC). Sections of samples
from the top, middle and bottom of each cylindrical cryogel
were analyzed. The results quoted in Table 1 indicate that
there are not statistical differences of both parameters VPT
and water content for the systems prepared with 1 or 2 wt
% of nanoparticles, respect to unloaded systems, reaching
values of hydration above 90 wt%. The presence of higher
amount of nanoparticles (5 wt%) seems to modify the VPT
parameter as a consequence of complex interactions with the
cryogel walls or even small aggregation of the nanoparticles.

Table 1. Hydration percentage (%H) obtained gravimetrically and VPT temperature of cryogels samples obtained by DSC.

Sample polyNIPA-CHI(0) polyNIPA-CHI(1) polyNIPA-CHI(2) ]polyNIPA-CHI(5)

H VPT H VPT H VPT H VPT

Zone [wt%] [8C] [wt%] [8C] [wt%] [8C] [wt%] [8C]

Top 93.0 32.0 92,9 31.5 92.2 30.7 85.4 30.9

Middle 93.3 31.2 93.8 30.7 91.1 32.6 95.6 37.2
Bottom 89.6 32.0 90.4 30.0 93.1 29.7 89.9 36.4
Mean 92.0
2.0 31.7 92.4
1.8 30.7 92.1
1.0 31.0 90.3
5.1 34.8

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Figure 8. NMR images of polyNIPA-CHI(0) cryogels (without nanoparticles) at 21 8C (left) and 36.5 8C (right) The zones used for the
determination of the signal intensities.

3.4.5. iNMR computed by iNMR as it is shown in Figure 8. [33] When

images are obtained using a Hahn spin echo, the Fourier
When a single fluid phase fills the pore space, the fluid transform of the measured signal, I(v,tE), is proportional to
content within a sample as a function of location can be the spin magnetization at the spin echo time, tE. The

Table 2. Apparent open porosity (Pi) polyNIPA-CHI cryogels at different temperatures calculated using Equation 3. Data normalized with
respect to sample polyNIPA-CHI(0) T. T, top; M, middle; B, bottom.

Pi [%]

Sample 21 8C 24 8C 28 8C 32 8C 34 8C 36.5 8C

polyNIPA-CHI(0) T 100.0 98.7 96.3 95.9 95.8 94.8

polyNIPA-CHI(0) M 97.3 96.3 94.1 94.0 91.7 89.8
polyNIPA-CHI(0) B 86.2 86.9 82.3 82.9 84.4 82.9
Mean 94.5
7.3 94.0
6.2 90.9
7.5 90.9
7.0 90.6
5.8 89.1
6.0
polyNIPA-CHI(1) T 90.2 91.2 92.1 86.4 86.7 86.6
polyNIPA-CHI(1) M 85.3 86.4 89.0 85.2 87.4 84.9
polyNIPA-CHI(1) B 85.3 87.6 84.9 79.9 79.4 78.8
Mean 86.9
2.8 88.4v2.5 88.6
3.6 83.9
3.4 84.5
4.4 83.4
4.1
polyNIPA-CHI(2) T 86.9 90.6 91.6 87.5 88.6 90.9
polyNIPA-CHI(2) M 91.1 95.4 96.4 92.4 91.1 91.4
polyNIPA-CHI(2) B 90.5 92.7 91.6 92.3 86.2 89.5
Mean 89.5
2.3 92.9
2.4 93.2
2.8 90.7
2.8 88.6
2.4 90.6
9.8
polyNIPA-CHI(5) T 93.8 93.0 92.2 90.4 90.3 92.0
polyNIPA-CHI(5) M 95.3 91.3 91.7 89.4 92.1 92.8
polyNIPA-CHI(5) B 82.2 80.4 80.0 79.8 79.4 77.1
Mean 90.4
7.2 88.2v6.8 88.0
6.9 86.6
5.8 87.3
6.9 87.3
5.1

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intrinsic signal intensity, I0(v), which corresponds to the

equilibrium magnetization (e.g., the signal obtained in the
limit tE ! 0), is proportional to the linear proton density,
which can be related to the porosity distribution of the
sample. Intrinsic magnetization cannot be measured, as the
equilibrium magnetization, which is oriented along the
direction of the static field, and it must be shifted onto the
orthogonal plane to be observed. However, I0(v) can be
estimated from a model of the relaxation process. The signal
corresponding to different degrees of magnetization
(obtained at different echo times) was measured, and the
intrinsic signal intensity (tE ¼ 0) was deduced estimating
the evolution of the signal intensity with echo time.[34]
The evolution of the signal intensity (Si) with the echo
time (tE) is shown in Figure 10 for the three selected
sample zones (top, middle, and bottom) of sample
polyNIPA-CHI(0). Figure 10. Temperature evolution of the cross-sectional area of
polyNIPA cryogels with different content of chitosan/bemiparin
The apparent open porosities (Pi) of the different cryogels
nanoparticles evaluated by image NMR: (&) polyNIPA-CHI(0); (~)
were calculated from the NMR signal intensities according polyNIPA-CHI(1); (*) polyNIPA-CHI(2); (*) polyNIPA-CHI(5).
to Equation 2 and the normalized results are reported in
Table 2. The porosity is very high, with values around 85–
90% for all the zones analyzed. The variation in the mean The NMR images of the cross-sections of sample
porosity of cryogels with temperature is not very high. polyNIPA-CHI(0) below and above the VPT shown in
However, the general trend is that the mean porosity Figure 8 reveal the decrease of the sample cross-sectional
decreases as the temperature is increased in the phase area as the cryogel is heated from bellow (left, low tE value,
transition region. At temperatures below the VPT of the corresponding to a temperature of 21 8C) to above (right,
cryogel, hydrogen bonding between hydrophilic segments high tE value, corresponding to a temperature of 36.5 8C) the
of the polymer chain and water prevails, leading to VPT. Figure 9 shows that the signal intensity decreases
enhanced swelling with the resulting network expansion. monotonically in a exponential way with the tE, at a given
Consequently, the pore size increases. As temperature temperature. The temperature evolution of the axial area of
increases, the polymer network seems to be contracted by polyNIPA-CHI cryogels with different nanoparticles con-
inter-polymer chain association through hydrophobic tent (0, 1, 2, and 5%) was measured and the results are
interactions, and part of the capillary bound water of the shown in Figure 10. The cross-sectional area of polyNIPA-
cryogel is expelled from the macropores, and the pore size CHI(0) cryogels presented a decrease of size with increasing
decreases.[35] temperature with a discontinuity in the vicinity of the VPT.
The VPT could be found by the intersection point of straight
lines described by the points at both sides of the curve. The
450000 values obtained by doing so ranged from 27.2 to 33.1 8C,
which are in the range of values determined by DSC
400000
experiments (see Table 1).
350000
Signal Intensity

300000
3.4.6. Bemiparin Release From Cryogels

Release of bemiparin from cryogels in PBS buffer (pH 7.4)

250000
was followed at 37 8C with the same procedure used for
200000 nanoparticles and the results are shown in Figure 11. By
comparison with Figure 3 it can be observed that bemiparin
150000
released from cryogels is lower than that released from free
100000
nanoparticles. This confirms the integration of the nano-
0 20 40 60 80 100 120 particles into the cryogel walls observed by SEM (Figure 7C).
T Echo Apparently, in this process the nanoparticles become
Figure 9. Evolution of the signal intensity versus TEcho for a covered by a chitosan layer that interacts with the
sample polyNIPA-CHI(0). A): top (*); middle (&); and bottom bemiparin modulating its release to the medium. The
(~). results obtained indicate that the bemiparin interacts

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0.25 4. Conclusion

0.2 Chitosan–bemiparin nanoparticles were obtained with

average sizes of 102
6.5 nm and a spherical morphology in
mg bemiparin released

which the bemiparin is affectively complexed with the

0.15
0.1
0.05
0
0 20 40
me (h)
Figure 11. Release profiles of bemiparin from chitosan/bemiparin
nanoparticles loaded cryogels in PBS, pH 7.4 at 37 8C. The
concentration of nanoparticles (in wt%) in the cryogels were
as follows: (&) 1%; (~) 2%; (*) 5%.
strongly with the cryogel matrix, which retains the
bioactive macromolecule in the site of the matrix. This
can be very interesting to have a local focalized effect with a
relative high and constant concentration of the bioactive
component in the site of application. Experiments in vivo
are in progress to demonstrate this focal effect.
3.4.7. MTT Test
The results from the MTT test are shown in Figure 12. All the
samples had the same behavior as the control, TMX, during
the 15 d of the experiment. This indicates that polyNIPA
cryogels and chitosan/bemiparin nanoparticles loaded
polyNIPA cryogels did not release cytotoxic leachable to
the medium.
chitosan. At pH 7.4 and pH 10, the bemiparin release is
1% sensitive to the pH. Bemiparin–chitosan nanoparticles
2% showed a similar biological activity than free bemiparin to
5% form the ternary complex with FGF2 and its membrane
receptor FGF 1c.
It was possible to synthesize polyNIPA-CHI cryogels
incorporating the chitosan/bemiparin nanoparticles,
60 80
according to results of SEM and FTIR-ATR spectroscopy.
The swelling kinetics of polyNIPA cryogels loaded with
chitosan/bemiparin nanoparticles at two temperatures
(above and below the VPT) was studied yielding an
expected behavior for this type of temperature dependent
system.iNMR and calorimetric techniques allowed us to
determine the morphology and properties of cryogels,
showing a very high porous material (>85%) with a VPT
between 30 and 35 8C. Both dimensions and porosity
decreased when temperature was raised, showing a
thermosensitive behavior due to the presence of PNIPA
in the cryogel walls. Loaded bemiparin nanoparticles were
well integrated into the porous walls using a dispersion of
the nanoparticles in a diluted chitosan solution, which
modulates the focal release of the bemiparin in the porous
of the cryogel matrix.
PolyNIPA cryogels and chitosan/bemiparin nanopar-
ticles loaded polyNIPA cryogels did not release cytotoxic
leachable to the medium and are therefore promising
materials for tissue engineering applications.
Acknowledgments: The authors thank Carlos Garc
ıa Aparicio for
the help with iNMR measurements. Financial support from CICYT-
MAT201018155 is gratefully acknowledged. Hazel Peniche thanks
the Agencia Espan ~ ola de Cooperacio

n Internacional para el
Desarrollo for the MAEC-AECID grant.

160 1 hour
Received: April 9, 2013; Revised: June 26, 2013; Published online:
2 hour DOI: 10.1002/mabi.201300184
140
1 day
2 days Keywords: bioactive bemiparin nanoparticles; image-NMR micro-
120
7 days structure characterization; macroporous polymeric systems;
100 15 days thermosensitive cryogels
cell viability %

80

60
[1] P. Perez, F. Plieva, A. Gallardo, J. S. Roman, M. R. Aguilar, I.
40
Morfin, Biomacromolecules 2008, 9, 66.
20 [2] V. I. Lozinsky, I. Y. Galaev, F. M. Plieva, I. N. Savina, H. Jungvid,
B. Mattiasson, Trends Biotechnol. 2003, 21, 445.
0
Termanox 0% 1% 2% 5% [3] A. Kumar, Eur. Cells Mater. 2008, 16, 11.
[4] A. Kumar, V. Bansal, K. S. Nandakumar, I. Y. Galaev, P. K.
Figure 12. Results of the MTT test for polyNIPA and chitosan/ Roychoudhury, R. Holmdahl, Biotechnol. Bioeng. 2006, 93,
bemiparin nanoparticles loaded polyNIPA cryogels together with 636.
the negative (TMX) control. Values are average
SD for n ¼ 15. [5] N. Bo €lgen, I. Vargel, P. Korkusuz, E. Gu
€ zel, F. Plieva, I. Galaev, J.
(p < 0.05) with respect to TMX. Biomed. Mater. Res. Part A 2009, 91, 60.

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201300184

www.MaterialsViews.com ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
11
R

Early View Publication; these are NOT the final page numbers, use DOI for citation !!

www.mbs-journal.de
[6] N. Kathuria, A. Tripathi, K. K. Kar, A. Kumar, Acta Biomater.
2009, 5, 406.
[7] Y. Hwang, N. Sangaj, S. Varghese, Tissue Eng. Part A. 2010, 16,
3033.
[8] M. Chalal, F. O. Ehrburger-Dolle, I. Morfin, J.-C. Vial, M.-R.
Aguilar de Armas, J. San Roman, N. Bo €lgen, E. PiSskin, O. Ziane,
R. Casalegno, Macromolecules 2009, 42, 2749.
[9] Y. Dogu, O. Okay, J. Appl. Polym. Sci. 2005, 99, 37.
[10] M. Biondi, F. Ungaro, F. Quaglia, P. A. Netti, Adv. Drug Delivery
Rev. 2008, 60, 229.
[11] A. G. Mikos, S. W. Herring, P. Ochareon, J. Elisseeff, H. H. Lu, R.
Kandel, Tissue Eng. 2006, 12, 3307.
[12] T. Matsumoto, D. J. Mooney, Adv. Biochem. Eng. Biotechnol.
2006, 102, 113.
[13] K. Rajangam, H. A. Behanna, M. J. Hui, X. Han, J. F. Hulvat, J. W.
Lomasney, Nano Lett. 2006, 6.
[14] M. R. Aguilar, L. Garc
ıa-Ferna
ndez, R. Palao-Suay, J. S. Roma
n,
in Heparin-Like Drugs with Antiangiogenic Activity (Ed: S.
Ran), InTech, Spain 2012.
[15] E. Da Pozzo, M. C. Barsotti, S. Bendinelli, A. Martelli, V. Calderone,
A. Balbarini, C. Martini, R. Di Stefano, Thromb. Res. 2012, 130,
e113.
[16] C. C. Lin, K. S. Anseth, Adv. Funct. Mater. 2009, 19, 23.
[17] C. Lin, P. D. Boyer, A. A. Aimetti, K. S. Anseth, J. Controlled
Release 2010, 142, 384.
[18] K. Norrby, APMIS 2006, 114, 79.
Macromol. Biosci. 2013, DOI: 10.1002/mabi.201300184
12 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
R
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
H. Peniche et al.
[19] Andrew K. Powell EAY, David G. Fernig, Jeremy E. Turnbull,
Glycobiology 2004, 14, 17R.
[20] A. Rapraeger, A. Krufka, B. Olwin, Science 1991, 252, 1705.
[21] H. Peniche, C. Peniche, Polym. Int. 2011, 60, 883.
[22] S. Buben
ıkova
, Mater. Sci. Forum 2007, 577, 539.
[23] F. Reyes-Ortega, G. Rodriguez, M. R. Aguilar, M. Lord, J.
Whitelock, M. H. Stenzel, J. Mater. Chem. B 2013.
[24] Chiou, Shu-Fen, Y. Chen, Biomaterials 2009, 30, 3332.
[25] M. Chen, H. Wong, K. Lin, H. Chen, S. Wey, K. Sonaje,
Biomaterials 2009, 30, 6629.
[26] C. Peniche, M. Ferna
ndez, A. Gallardo, A. Lo

pez-Bravo, J. S.
Roma
n, Macromol. Biosci. 2003, 3, 540.
[27] A. Nilasaroya, L. A. Poole-Warren, J. M. Whitelock, P. Jo
Martens, Biomaterials 2008, 29, 4658.
[28] I. Capila, R. J. Linhardt, Angew. Chem. Int. Ed. 2002, 41, 390.
[29] M. Marcos, P. Cano, P. Fantazzini, C. Garavaglia, S. Gomez, L.
Garrido, Magn. Reson. Imag. 2006, 24, 89.
[30] A. Domard, Int. J. Biol. Macromol. 1987, 9, 98.
[31] A. Denuziere, D. Ferrier, O. Damour, A. Domard, Biomaterials
1998, 19, 1275.
[32] N. I. Sushko, S. P. Firsov, R. G. Zhbankov, V. M. Tsarenkov, M.
Marchewka, C. Ratajczakc, J. Appl. Spectrosc. 1994, 61, 704.
[33] R. Kulkarni, A. T. Watson, AIChE J. 1997, 43, 2137.
[34] A. T. Watson, C. T. P. Chang, Progr. Nucl. Magn. Reson.
Spectrosc. 1997, 31, 343.
[35] Y. Qiu, K. Park, Adv. Drug Delivery Rev. 2001, 53, 321.
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