Materials Sciences and Applicatio ns, 2011, 2, 509-520
doi:10.4236/msa.2011.26069 Published Online June 2011 (http://www.SciRP.org/journal/msa)
509
Interpenetrated Chitosan-Poly(Acrylic
Acid-Co-Acrylamide) Hydrogels. Synthesis,
Characterization and Sustained Protein
Release Studies
Michel Bocourt Povea1, Waldo Argüelles Monal2, Juan Valerio Cauich Rodríguez3,
Alejando May Pat3, Nancy Badas Rivero1, Carlos Peniche Covas1
1Department of Macromolecular Chemistry, University of Havana, Havana, Cuba; 2Center for Food and Development Research, Unit
Guaymas, Sonora, México; 3Scientific Research Centre of Yucatan, Mérida, México.
Received March 1st, 2011; revised April 16th, 2011; accepted May 5th, 2011.
ABSTRACT
Interpenetrated polymer networks of chitosan (CHI), polyacrylic acid (PAA) and polyacrylamide (PAM) were prepared
by free radical polymerization. These hydrogels were either washed with double distilled water (CHI/PAA/PAM) A or
hydrolyzed with 1M sodium hydroxide (NaOH), (CHI/PAA/PAM) S. Both types of hydrogels were characterized by in-
frared spectroscopy, microstructural techniques and compressive mechanical testing. Finally, hydrogels were loaded
with bovine serum albumin (BSA) and release followed at different pHs. Infrared spectra analysis showed correspon-
dence between hydrogels and monomer feed compositions. Hydrolyzed hydrogels, had increased water content and pH
swelling dependence. Compression modulus of swelled hydrolyzed hydrogels decreased with increasing equilibrium
water content. Higher BSA loadings were achieved on hydrolyzed hydrogels due to their high water content and poros-
ity. Protein release from hydrogels was low ( 20% after 10 hours) at pH 1.2, but sustained release was observed at pH
6.8 and 7.4. The integrity of the protein released at 6.8 and 7.4 by hydrolyzed hydrogels was unaffected. The hydrogles
showed no cytotoxic effects on human skin dermal fibroblasts as determined by MTT assay except for two compositions
of (CHI/PAA/PAM) A samples, which after seven days presented a viability lower than 80% respect to the control.
Keywords: Hydrogels, Controlled Release, Protein, Polyacrylamide, Interpenetrated Polymer
1. Introduction
Hydrogels are three dimensional hydrophilic polymer
networks that swell, but do not dissolve, when brought
into contact with water [1]. Hydrogels have been actively
studied, particularly those experiencing reversible vol-
ume changes in response to external stimulus, such as pH,
temperature and ionic concentration. These “smart” hy-
drogels have found applications in biomedicine and bio-
technology [2] including soft contact lenses [3], immobi-
lization of enzymes and proteins [4], antibodies and an-
tigens [5] and matrices for drug delivery systems [6,7].
The ability of these hydrogels to respond to their envi-
ronment increase drug loading and provide protection
from environmental conditions such as those found in the
gastrointestinal tract [8]. In this regard, stimuli responsive
hydrogels can be useful for the design of site-specific
drug delivery devices; for instance, colon-specific drug
delivery systems. Another important advantage of these
hydrogels is that the active ingredient remains on the
organ or tissue for longer times than conventional ones
[9].
Intelligent hydrogels have also been prepared from in-
terpenetrated polymer networks (IPNs), and it has been
found that they combine high swelling capacity with in-
creased mechanical properties [10,11]. Superabsorbent
hydrogels of poly(acrylamide-co-acrylic acid) character-
ized mainly by fast swelling and pH swelling dependence,
ionic strength and composition have been prepared by a
number of authors [12-14]. These materials have been
proposed for different applications, such as waste water
purification from textile industry [15], for the environ-
mental analysis of Cu and Cd [16] and as mechanical

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 510
Characterization and Sustained Protein Release Studies
transducers [17].
A great variety of systems have been prepared by graft-
ing synthetic polymers onto polysaccharides. Of particu-
lar interest are those based on the graft polymerization of
monomers such as acryl amide [18], acrylic acid [19],
N-isopropylacrylamide [20] and N-vinylpyrrolidone [21]
onto chitosan. Chitosan, (1,4)-[2-amino-2-deoxy-β-D-
glucan] is a deacetylated derivate of chitin, a natural oc-
curring polymer. It is widely studied in pharmaceutical
and biomedical fields because of its biodegradability,
biocompatibility, non toxicity and interesting structural
properties [22]. The functional amino and hydroxyl
groups in chitosan allow the preparation of chitosan de-
rivatives with improved properties under relatively mild
conditions [23]. Mahdavinia et al. synthesized poly (ac-
rylic acid-co-acrylamide) grafted chitosan, and studied
the effect of the buffer solution, ionic strength and cro-
ss-linker concentration on the swelling properties of
these systems. They observed that water uptake increased
with increasing the acrylic acid concentration in the mo-
nomer feed. However after hydrolysis with 1 N NaOH at
100˚C for 60 min the samples with high acrylamide ratio
exhibited increased swelling [24]. However, in this study
no use was made of microstructural techniques (EDX,
SEM) for characterizing the hydrogels prepared and there
was no report on their mechanical properties, biocom-
patibility and their potential as matrices for drug or pro-
tein release.
In the present work we prepare chitosan-poly(acrylic
acid-co-acrylamide) IPNs by the free radical co-polym-
erization of acrylamide and acrylic acid in the presence
of chitosan and methylenebisacrylamide as a cross-linker.
Chitosan concentration in the feed was kept constant and
low in all preparations so as to produce hydrogels with
increased pH sensitivity. The effect of monomer feed
composition (either acrylic acid or acrylamide) and alka-
line hydrolysis was studied not only in terms on mor-
phology and water uptake but also in terms of their com-
pressive properties. Hydrogel cytotoxicity was assessed
by determining viability of cells by the MTT test. Bovine
Serum Albumin (BSA) was used as model protein to
study their drug release properties. Finally, the effect of
pH on protein release kinetics was also studied.
2. Experimental
2.1. Materials
Chitosan (CHI; D.D. = 90% determined by potentiometry,
Mv = 9.3 × 104) was purchased from Primex (Iceland).
Acrylic acid (AA, Aldrich, Milwaukee, WI) was distilled
under reduced pressure before use. Acrylamide (AM;
Fluka, Buchs, Switzerland), Ammonium persulfate (APS;
Fluka, Buchs, Switzerland), N,N-methylenebisacrylamide
(MBA; Sigma Chemical Co., St. Louis, USA,), N,N,N,N
tetramethylenediamine (TEMED; Fluka, Buchs, Swit-
zerland) and bovine serum albumin (BSA; Sigma-Al-
drich, Zwijndrecht, Netherlands), Mw = 6.5000 × 104)
were used as supplied. All other reagents were of ana-
lytical grade and used as received. Double distilled water
was used through this work.
2.2. Methods
2.2.1. Preparation of (CHI/PAA/PAM) Hydrogel
Hydrogels were prepared by free radical polymerization
of AM and AA in presence of the CHI. A determined
mass of AM and AA, together with MBA (3.3 × 10–2 mol/L),
APS (1.2 × 10–3 mol/L) and TEMED in a 1:1 molar ratio
with APS, were added to a 1% chitosan solution in 1%
acetic acid and stirred for 10 minutes at room tempera-
ture. The solution was placed in a glass tube, degassed
and sealed in vacuum. Finally the glass tube was placed
in a water bath at 50˚C for 24 h. These hydrogels will be
referred to as M1-M6 and their composition is listed in
Table 1.
The hydrogels obtained in long cylindrical shapes
were sliced into discs of 2 cm diameter 2 mm thick.
Some hydrogels were washed in double distilled water
for approximately 2 weeks to remove unreacted mono-
mer and are named (CHI/PAA/PAM)A and others were
hydrolysed with 1 mol/L sodium hydroxide solution,
during 3 hours and washed with double distilled water
until pH 7 was reached. These are identified as (CHI/
PAA/PAM)S. The discs were dried to constant weight at
40˚C in a vacuum oven.
2.2.2. FTIR Analysis
FTIR spectra were obtained with KBr discs and recorded
in the spectral range from 4000 to 400 cm–1 by using a
Nicolet model Protege, 460 spectrometer (Nicolet In-
Table 1. Relative composition of the reaction mixtures for
the preparation of (CHI/PAA/PAM) systems.
SAMPLESCHI (g)AA (g) AM (g)
Monomer feed ratio
CHI/AA/AM
(wt%)
M1 0.08 0.00 0.72 10/0/90
M2 0.08 0.08 0.64 10/10/80
M3 0.08 0.16 0.56 10/20/70
M4 0.08 0.36 0.36 10/45/45
M5 0.08 0.56 0.16 10/70/20
M6 0.08 0.72 0.00 10/90/0

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 511
Characterization and Sustained Protein Release Studies
strument Corp., Madison, WI). Spectra were obtained
with a resolution of 2 cm–1 and were averaged over 100
scans.
2.2.3. Swelling Studies
The discs were cut into fragments of about 30 mg and
weighed accurately. These fragments were placed into
flasks with 10 mL solution of a given pH and kept in a
thermostated bath at 37˚C. Solutions with pH 1.2 (simu-
lated gastric fluid), pH 7.4 (phosphate buffered saline),
and pH 6.8 (double distilled water) were prepared. The
water uptake, W, was calculated by measuring the weight
gain of the sample at different times after carefully wip-
ing the surface with a filter paper. It was reported as
0
0
t
WW
WW

 (1)
where W0 is the weight of the dry sample and Wt is that of
the sample at time t.
2.2.4. Scanning Electron Microscopy (SEM)
The morphology of the hydrogels was determined using
a scanning electron microscope (SEM-JEOL JSM-
6360LV, Japan). Small pieces of swelled gels were
freeze-dried to avoid the collapse of porous structure.
Then they were cut with a knife to expose the inner sur-
face. Samples were placed on an aluminum mount, sput-
tered with gold palladium, and then scanned at an accel-
erating voltage of 15 kV.
2.2.5. Mechanical Properties
For the determination of compressive properties, fully
swollen hydrogels were cut into cylindrical species of 6
mm diameter and 12 mm length measured with a Mitu-
toyo digital caliper. Hydrogels were confined in a cylin-
drical metal frame and compressed at 0.5 mm/min, using
a Minimat testing machine with a 10 N load cell. The
stress (σ) and strain (ε) were obtained according to Equa-
tions (2) and (3).
P
A
  (2)
0
F
l
l
  (3)
where P represents the compression force, A the area of
the cylinder and l0 and lF are the initial and final length,
respectively. From the initial portion of the stress-strain
curve, the modulus was obtained and the values reported
as the mean ± SD of six determinations.
2.2.6. Elemental Analysis
Elemental analysis (C, N, O and Na) was conducted with
an INCA 7582 Elemental Microanalyzer (Oxford In-
struments, UK) coupled to the Jeol JSM-6360LV SEM.
2.2.7. BSA Release Studies
2.2.7.1. BSA Loading
Dried sample discs weighing 30 mg were placed in vials
containing 10 mL of aqueous solutions of BSA (1.0 mg/
mL). Then, vials were placed in a temperature–regulated
incubator at 4˚C. After 7 days, hydrogels were separated
from the solutions and freeze-dried. The concentration of
the supernatant solutions were analyzed with the modi-
fied Coomassie Blue protein assay (Biorad) using UV
spectroscopy at 595 to determine the amount of BSA
loaded in each hydrogel disc [25]. The BSA load in hy-
drogels was determined from the difference in the solu-
tions concentrations before and after incubation. All ex-
periments were performed in triplicate.
2.2.7.2. In Vitro Protein Release Study
Release of BSA was carried out as established by the
USP XXIV Standard. Three different release media were
tested: simulated gastric fluid (SGF), consisting of NaCl
(2.0 g), HCl (7 mL) and pH adjusted to 1.2 ± 0.5; simu-
lated intestinal fluid (SIF), consisting of KH2PO4 (6.8 g),
0.2 N NaOH (77 mL), and pH adjusted to 6.8 ± 0.1.
Phosphate buffered saline (PBS) 7.4 was prepared with
deionized water. For release studies the BSA–loaded
hydrogel discs were suspended in 10 mL of these solu-
tions at 37˚C in a ZHICHENG ZHWX-200B Incubator
with reciprocating motion (100 rpm). At predetermined
time intervals, 200 µl aliquots were removed and simul-
taneously replenished by 200 µl of fresh solutions to
maintain sink conditions. Protein content of each sample
was analyzed with the modified Coomassie Blue protein
assay (Biorad) using UV spectroscopy at 595 nm. A ca-
libration curve was generated at each time interval using
a non-loaded gel in order to correct for the intrinsic ab-
sorbance of the polymer. All experiments were per-
formed in triplicate.
2.2.7.3. Analysis of Released Proteins by Gel
Electrophoresis (SDS-PAGE)
The structural integrity of BSA released from CHI/PAA/
PAM) hydrogels was analyzed by SDS-PAGE in 10%
acrylamide gels according to Laemli [26] using a Mini-
Protean II Cell (Bio-Rad Laboratories Hercules, CA,
USA). BSA solutions were directly loaded into the test
wells with a micropipette, and the electrophoresis was
performed at 100 V. The gel was stained with 0.1%
AgNO3 to visualize protein bands. The study was con-
ducted according to the manufacturer’s protocol. The gel
pictures were taken with a scanner after wiping off all the
water from the gel membrane.

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 512
Characterization and Sustained Protein Release Studies
2.3. Cytotoxicity Test
2.3.1. In Vitro Cell Culture for Biocompatibility
Experiment
Sterile hydrogel pieces M1-M6 (CHI/PAA/PAM)A and
M1-M6 (CHI/PAA/PAM)S of uniform weight (50 mg
each) were used for biocompatibility experiments. The
negative control was tissue culture plastic, Thermanox
(TMX), in discs of 13 mm diameter (Nunc) and the posi-
tive control (toxic agent) was Triton-1% (Merck). Ex-
periments were carried out using human skin dermal fi-
broblasts (Pharmakine DPK-SKDF-H). Cells were cul-
tured at 37˚C and 5% CO2. The culture medium was
Dulbecco’s modified Eagle’s medium (DMEM), modi-
fied with HEPES (Sigma), rich in glucose and supple-
mented with 10% (v/v) fetal bovine serum (FBS; Gibco),
200 mM glutamine (Sigma), 1% of penicillin-streptomycin
solution (10000 Units/mL penicillin and 10 mg/mL
streptomycin, Sigma). The culture medium was changed
at selected time intervals with care to cause little distur-
bance to culture conditions.
2.3.2. MTT Assay
TMX, M1-M6 (CHI/PAA/PAM)A and M1-M6 (CHI/
PAA/PAM)S discs were set up in 5 mL of DMEM. They
were placed on a roller mixer at 37˚C and the medium
was removed at different time periods (8 h, 1, 4, 7 days)
[27] and replaced with other 5 mL of fresh medium. All
the extracts were obtained under sterile conditions. Fi-
broblasts cells were seeded at a density of 105 cells/mL
in complete medium in a sterile 96-well culture plate and
incubated to confluence. Then, the medium was replaced
with the corresponding eluted extract and incubated at
37˚C for 24 h. A solution of MTT was prepared in warm
PBS (0.5 mg/mL; Sigma) and the plates were incubated
at 37˚C for 4 h. Excess medium and MTT were removed
and 100 μl dimethylsulphoxide (DMSO; Sharlau) was
added to all wells in order to dissolve the MTT taken up
by the cells. This was mixed for 10 min and the absorb-
ance was measured with a Biotek SYNERGY spectro-
photometer using a test wave length of 570 nm and a
reference wave length of 630 nm. From the values of
relative cell viability a statistical analysis was carried out
using Student t test in order to detect significant differ-
ences between the distribution of measured values for the
negative control and those obtained for the experimental
groups studied.
3. Results and Discussion
3.1. Synthesis and Spectral Characterization
The free radical polymerization of AA and AM in the
presence of chitosan results in a hydrogel in which chi-
tosan chains become grafted with the copolymer [19,24].
The use of the difunctional monomer MBA in the react-
ing mixture promotes the formation of a tridimensional
interpenetrated polymer network reinforcing the me-
chanical properties of the hydrogel as well as restraining
its swelling capacity [28]. During the alkaline treatment,
amide groups are hydrolyzed into carboxylate ions. The
FTIR spectra of CHI, AM, AA, (CHI/PAA/PAM)A and
(CHI/PAA/PAM)S for composition corresponding to M5
are shown in Figure 1(A).
The spectra of hydrogels at other compositions were
qualitatively similar to that of M5 and are not shown.
Chitosan spectrum exhibited the distinctive absorption
bands at 1658 cm–1 (Amide I), 1595 cm–1 (–NH2 bending)
and 1314 cm–1 (Amide III). The absorption bands at
1154 cm–1 (anti-symmetric stretching of the C–O–C bri-
dge), 1082 and 1032 cm–1 (skeletal vibrations involving
the C–O stretching) are characteristic of its saccharide
structure [29].
The IR spectrum of PAA exhibits the characteristic
absorption band at 1728 cm–1 due to the C=O stretching
vibration of the carboxylic groups. The intense band at
1670 cm–1 in the spectrum of PAM corresponds to the
C=O stretching vibration (Amide I). The main absorption
bands of the CHI, AM, AA infrared spectra, appeared
also in (CHI/PAA/PAM)A and (CHI/PAA/PAM)S spec-
tra. The bands at 1558 cm–1 (asymmetric COO– stretch-
ing) and 1406 cm–1 (symmetric COO– stretching) present
in the (CHI/PAA/PAM) S spectrum result from the alka-
line hydrolysis of the amide groups of AM into carboxy-
late ions.
Since the strong absorption band at 1082 cm–1 in the
chitosan spectrum is absent in the spectrum of PAM, the
ratio of the intensity of the absorption band at 1630 cm–1
(A1630) to the intensity of the absorption band at 1082
cm–1 (A1082) was used as indicative of the composition of
the sample. As it can be seen in Figure 1(B) the absorp-
tion bands ratio A1670/A1082 is very sensitive to the AM
composition of the systems i.e. it decreased when the
concentration of AA increased in the reaction mixture.
This trend indicates that the proportion of AA in the hy-
drogels increased from M1 to M6, as expected from the
monomer feed composition. Furthermore, the values of
the ratio A1670/A1082 for (CHI/PAA/PAM)S hydrogels
were smaller than those of the corresponding (CHI/PAA/
PAM)A samples indicating that hydrolysis of the amide
groups on PAM to carboxylate ions was effectively
achieved by the treatment with NaOH.
EDX analysis of hydrogels corroborated the IR spec-
troscopy results. Figures 2(a) and (b) show the EDX of
the sample M4 (CHI/PAA/PAM)A and M4 (CHI/PAA/
PAM)S, respectively, which are representative of the

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 513
Characterization and Sustained Protein Release Studies
(A)
(B)
Figure 1. (A): FTIR spectra of (a), CHI; (b), M5(CHI/PAA/
PAM)S; (c) M5(CHI/PAA/PAM)A; (d) PAM and (e) PAA.
(B): Absorption bands ratio A1670/A1082 of (●), (CHI/PAA/
PAM)A and (○), (CHI/PAA/PAM)S hydrogels as function of
the initial acrylic acid composition in the polymerization
mixture.
corresponding series.
The peak at 1.05 keV assigned to the presence of so-
dium (Na) was observed in Figure 2(b) and attributed to
the formation of sodium carboxylate salts resulting from
the basic hydrolysis of acrylamide moieties.
(a)
(b)
Figure 2. EDX analysis of hydrogels (a): M4(CHI/PAA/
PAM)A, (b): M4(CHI/PAA/PAM)S.
3.2. Swelling of Hydrogels
Swelling kinetics of (CHI/PAA/PAM)A and (CHI/PAA/
PAM)S hydrogels in water has been reported before Bo-
court et al. [30]. In general, swelling was fast in both
cases, although the rate of water uptake in hydrolyzed
samples was always higher than the corresponding
non-hydrolyzed ones. The process followed a second
order kinetics with respect to the remnant swelling. This
behavior is typical of processes in which solvent diffu-
sion is controlled by the relaxation of chains [31]. Water
swelling at equilibrium of (CHI/PAA/PAM)A and (CHI/
PAA/PAM)S hydrogels prepared at different monomer
feed ratios are shown in Figure 3.
As expected, swelling is strongly dependent on com-
position, since the hydrophilic character of the functional
groups provided by the structural units in the hydrogels is

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis,
Characterization and Sustained Protein Release Studies
514
resulting from the basic hydrolysis of amide groups of
acrylamide units. These negatively charged carboxylate
ions are highly solvated and the repulsion between them
provoke the expansion of the hydrogel network absorb-
ing more water than the neutral amide groups. Although
both factors increase swelling, this effect is more notice-
able on M1 samples whose composition is the richest in
PAM. As the AM/AA ratio decreases from M2 to M5
less water is absorbed, although swelling was always
higher than that of the corresponding (CHI/PAA/PAM)A
hydrogels. A point to note is that sample M6 exhibited a
significantly higher swelling compared to samples M4
and M5. Apparently in the latter two there are still some
interactions between non-hydrolised AM units and sev-
eral non-dissociated carboxylic groups of AA. The pH
dependence of the swelling capacity of hydrogels was
evaluated at pH 1.2 and pH 7.4 and the results are re-
ported in Table 2.
Figure 3. Equilibrium (ultimate) swelling in water of (●),
(CHI/PAA/PAM)A and (○), CHI/PAA/PAM)S hydrogels as
function of the initial acrylic acid composition in the po-
lymerization mixture.
not the same, but varied in the order: –COO– > –COOH >
–CONH2 > –OH. In addition, interactions between func-
tional units can affect swelling. For instance, swelling of
(CHI/PAA/PAM)A hydrogels decrease as the AM/AA
ratio decreases, due to the formation of hydrogen bond-
ing between carboxylic groups in PAA and amide groups
in AM. These hydrogen bonds introduce additional
cross-links to the hydrogel network decreasing its swell-
ing capacity. Complexation of PAA with PAM and
poly(AA-co-AM) has been reported by K. Sivadasan et
al. [32] using pyrene labeled polymers.
Swelling of (CHI/PAA/PAM)A hydrogels (samples
M2 to M6) is higher at pH 7.4 than at pH 1.2 due to the
dissociation of the carboxylic units of AA above pH 4.7
(pKa = 4.7). However, no significant change in swelling
with pH was observed for sample M1, due to the neutral
character of acrylamide. At pH 1.2 the free amino groups
of chitosan are protonated, but its proportion in M1 is so
low that its effect on swelling is negligible. Similar be-
havior is observed for (CHI/PAA/PAM)S hydrogels
(samples M1 to M5). In this case sample M1 also exhibit
swelling dependence on pH since some of its neutral
amide groups have been hydrolyzed. On the other hand,
swelling of sample M6(CHI/PAA/PAM)S at pH 1.2 and
7.4 do not differ significantly with swelling of sample
M6(CHI/PAA/PAM)A since their composition was not
altered by the alkaline treatment.
In the absence of AM (sample M6) there is a further
decrease in swelling which should be attributed to the
formation of interpolyelectrolyte salt bonds between the
amino groups of chitosan and the carboxylic groups of
AA moieties. This will introduce additional cross-links to
the network, with the consequent decrease in swelling.
(CHI/PAA/PAM)S hydrogels have higher swelling ca-
pacity than the (CHI/PAA/PAM)A hydrogels (Figure 3)
as a consequence of the formation of carboxylate ions
The effect of ionic strength on (CHI/PAA/PAM) hy-
drogels swelling can be appreciated by comparing the
swelling degrees of (CHI/AA/AM)S hydrogels at pH 7.4
(see Table 2) with those achieved in water (see Figure 3).
Table 2. Swelling capacity of (CHI/PAA/PAM)A and (CHI/PAA/PAM)S at different pH.
(CHI/AA/AM)A (CHI/AA/AM)S
Samples CHI/AA/AM (wt%)
pH 1.2 pH 7.4 pH 1.2 pH 7.4
M1 10/0/90 14.0 ± 0.7 13.2 ± 0.1 11.5 ± 0.9 16.3 ± 0.7
M2 10/10/80 10.3 ±0.3 21.5 ± 1.3 6.7 ± 0.7 26.0 ± 1.6
M3 10/20/70 6.9 ± 0.4 23.2 ± 1.0 4.2 ± 0.2 27.5 ± 1.4
M4 10/45/45 3.9 ±0.2 24.6 ± 1.5 2.4 ± 0.1 28.1 ± 1.4
M5 10/70/20 2.9 ± 0.1 24.9 ± 1.7 2.1 ± 0.1 28.6 ± 1.9
M6 10/90/0 1.6 ± 0.1 29.8 ± 1.9 1.5 ± 0.1 32.5 ± 1.6

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 515
Characterization and Sustained Protein Release Studies
This occurs because the ionic strength provided by the
phosphate buffered saline (I = 0.2) hampers swelling in
spite of the higher dissociation degree expected for the
carboxylic groups of AA at pH 7.4. In this case, charge
screening of the counterions on the fixed charges of the
polymer network, leads to a decrease in the osmotic
pressure [33].
3.3. Morphologic Studies
Figure 4 shows the SEM pictures of M4(CHI/PAA/
PAM)A and M4(CHI/PAA/PAM)S hydrogels. From
these pictures, the higher porosity of the hydrolyzed
sample is evident. The same morphologies were ob-
served for hydrogels prepared at other compositions.
This increased porosity allows faster water diffusion
through the hydrogel network which in turn is another
factor that contributes to the higher rate of water uptake
observed in hydrolyzed samples. It is important to men-
tion that even when elimination of water by different
means leads to different structures, in this case the same
method was used for all hydrogels.
(a)
(b)
Figure 4. SEM pictures of M4 hydrogels: (a) M4(CHI/
PAA/PAM)A and (b) (CHI/PAA/PAM)S.
3.4. Mechanical Properties
Hydrogels can swell to several hundred times within a
few minutes. However, the modulus of hydrogels will
decrease when decreasing polymer fraction, as predicted
by rubber elasticity theory. Therefore, highly swelled
hydrogels are usually mechanically poor and difficult to
handle without breaking. This is an obvious limitation for
applications requiring mechanical performance like soft
tissue replacement and addition to drug release matrices.
In relation to this, the preparation of semi-interpenetrated
or interpenetrated polymer networks is a suitable proce-
dure to achieve superabsorbent hydrogels with better
mechanical properties. The improvement of the me-
chanical properties of IPN hydrogels is due to an increase
in polymer mass as well as inter- and intra-molecular
interactions from the incorporation of the second network
into the normal hydrogel network. The initial compres-
sion modulus of (CHI/PAA/PAM)S systems with differ-
ent composition at equilibrium swelling are shown in
Table 3.
As expected, the compression modulus decreases in
the sequence M5  M4  M3  M6  M2  M1, i.e. by
increasing hydrogel water content. The values of the
compression moduli found for (CHI/PAA/PAM)S hy-
drogels are of the same order of magnitude as those re-
ported by Tang et al. [34] for high mechanical strength
(PAA/PAM) IPN hydrogels.
3.5. BSA Incorporation and in Vitro Protein
Release
Therapeutic protein administration usually faces the need
of frequent doses due to the small residence time in
blood of the protein. A promising strategy to overcome
this drawback is the development of sustained drug re-
lease systems based on hydrogel matrices loaded with the
therapeutic drug [35].
Loading capacities for BSA of (QUI/PAA/PAM)A and
(QUI/PAA/PAM)S are reported in Table 4, together with
Table 3. Compression modulus and swelling capacity in
water of (CHI/PAA/PAM)S hydrogels.
SAMPLES COMPRESSION
MODULUS (kPa)
SWELLING
(g H2O/g polymer)
M1 45 ± 2 819 ± 45
M2 68 ± 2 246 ± 16
M3 84 ± 3 216 ±11
M4 110 ± 7 99 ± 8
M5 122 ± 9 86 ± 2
M6 76 ± 4 231 ± 16

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 516
Characterization and Sustained Protein Release Studies
Table 4. Protein load and swelling of (CHI/PAA/PAM)A and (CHI/PAA/PAM)S hydrogels in 1 mg/mL BSA solution.
(CHI/PAA/PAM)A (CHI/PAA/PAM)S
Samples Load (mg/g) Swelling
(g H2O/g polymer) Load (mg/g) Swelling
(g H2O/g polymer)
M1 232.2 25.7 511.3 677.8
M2 202.2 20.8 618.3 210.7
M3 141.9 15.3 730.4 166.5
M4 0 4.2 323.6 72.5
M5 0 3.1 200.6 63.8
M6 0 1.9 172.7 185.6
the water uptake of hydrogels in these experimental con-
ditions. In the case of (QUI/PAA/PAM)A hydrogels only
samples M1 to M3 were able to incorporate the protein.
Apparently, both low swelling and small pore sizes on
samples M4 to M6 did not allow BSA incorporation.
On the other hand, highly swollen (QUI/PAA/PAM)S
hydrogels incorporated BSA at all compositions. Higher
loadings were achieved for samples with lower AA con-
tent (M1 to M3) while samples M4 and M5 which are
richer in AA content, exhibited lower swelling. The
cause of this should be the higher density of carboxylate
groups these samples should posses in the surface, which
could hamper BSA adsorption by anionic electrostatic
repulsion between the protein and the hydrogel surface.
This explains why sample M6 (which is essentially a
CHI/PAA hydrogel) having a similar swelling degree as
sample M3 exhibits only 24% its BSA loading capacity.
A similar effect was reported by Karadag et al. [36] for
BSA adsorption on acrylamide-itaconic acid hydrogels.
BSA release from (CHI/PAA/PAM) hydrogels was
studied at three different pHs: pH 1.2 (SGF); pH 6.8,
(SIF), and pH 7.4 (PBS). Figure 5 shows the in vitro
cumulative release profiles of the BSA loaded (CHI/
PAA/PAM)A hydrogels. At pH 1.2 protein release is
favored in AM richer samples (M1 > M2 > M3), since in
acid medium the hydrogels containing AA units become
more compact due to hydrogen bonding of carboxylic
groups of AA with the amide groups of AM units.
However, a considerable amount of BSA remains
trapped in the gels at pH 1.2, presumably due to interac-
tions between the protein and the polymer. At pH 6.8 and
7.4 the carboxylic groups of AA are dissociated (pKa =
4.7), and the consequent expansion of the gels together
with the electrostatic repulsive force between the (COO–)
charged sites of AA and BSA (pK = 4.8) favours protein
release, reaching 80 per cent at pH 6.8 and almost 100
per cent at H 7.4. It is worth pointing out that BSA re-
lease form (CHI/PAA/PAM) hydrogels was relatively
fast at pH 6.8 and 7.4 because after 10 hours it reached
almost 100 per cent.
On the other hand, BSA release from (QUI/PAA/
PAM)S hydrogels (Figure 6) at pH 1.2 was even lower
than that of (CHI/PAA/PAM)A samples (about 20 per
cent for all compositions).
This is to be expected since at this pH they should be
more compact due to poorer swelling (see Table 2) than
(CHI/PAA/PAM)A hydrogels. At pH 6.8 and 7.4 protein
release resulted slower and more composition dependent
for (QUI/PAA/PAM)S than for (QUI/PAA/PAM)A hy-
drogels. BSA release increased with increasing AA con-
tent (M1 < M2 < M3 < M4 < M5 < M6). Best release
profiles were obtained at pH 7.4 for samples M4 to M6,
which after liberating approximately 20% of the protein
during the first hour exhibited sustained release for the
48 hours of the experiment.
3.6. BSA Structural Integrity
The effect of the loading-release process on the integrity
of BSA was studied, since it may have affected the pro-
tein structure and stability. Possible detrimental effects
include protein denaturation, aggregation and hydrolysis.
Experiments were carried out on the BSA released from
(QUI/PAA/PAM)A and (QUI/PAA/PAM)S hydrogels
after 2 days and compared with the original BSA in solu-
tion (a BSA standard).
Figure 7 shows the SDS-PAGE results of the released
and commercial BSA. Molecular weight markers are
shown in lane I. The SDS-PAGE gel banding patterns of
the BSA released from the (QUI/PAA/PAM)A hydrogels
at pH 1.2, 6.8 and 7.4 are shown in lanes II, III and IV
respectively. Similarly the protein released from the
(QUI/PAA/PAM)S hydrogels at pH 1.2, 6.7 and 7.4 ap-
pear in lanes V, VI and VII, respectively and the com-
mercial BSA, lane VIII, exhibited clear bands at 66 KDa.
The presence of bands of lower molecular weight species
in lanes II, III and V indicates appreciable modification

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 517
Characterization and Sustained Protein Release Studies
Figure 5. In vitro cumulative release profiles of BSA from (CHI/PAA/PAM)A hydrogels at various pH. (■) M1; (●) M2; (▲)
M3.
Figure 6. In vitro cumulative release profiles of BSA from (CHI/PAA/PAM)S hydrogels at various pH. (■) M1; (●) M2; (▲)
M3; (▼) M4; (◄) M5; (►) M6.
Figure 7. SDS-PAGE analysis of BSA. Lane I corresponds molecular weight markers. Lanes II, III, IV correspond BSA re-
leased from (QUI/PAA/PAM)A hydrogels at pHs 1.2, 6.8 and 7.4 respectively. Lanes V, VI, VII correspond BSA released
from (QUI/PAA/PAM)S hydrogels at pHs 1.2, 6.8 and 7.4 respectively. Lane VIII correspond BSA standard.
the released protein. On the other hand, lanes IV, VI and
VII indicate that BSA released from (QUI/PAA/PAM)A
hydrogels at pH 7.4 and (QUI/PAA/PAM)S hydrogels at
pH 6.7 and 7.4 maintain its structural integrity, which is a
premise for preserving the protein activity.
3.7. Cytotoxicity Test
Hidrogel cytotoxicity was assessed by determining vi-
ability of cells by the MTT assay. Viability of human
skin dermal fibroblast exposed to hydrogel extract is
shown in Figures 8 and 9. For (QUI/PAA/PAM) hy-
drogels at different times. All the samples have the same
behavior than the control Thermanox (TMX), resulting
not cytotoxic, except for M1 and M6 (QUI/PAA/PAM)A
at 7 days which presents a viability below 80% with re-
pect to the control. s

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 518
Characterization and Sustained Protein Release Studies
Figure 8. Results of MTT test for M1-M6 (CHI/PAA/PAM)A discs. Values are average ± standard deviation for n = 8. *p <
0.05 and ***p < 0.001 with respect to TMX.
Figure 9. Results of MTT test for M1-M6 (CHI/PAA/PAM)S discs. Values are average ± standard deviation for n = 8. *p <
0.05 and ***p < 0.001 with respect to TMX.
This could be related with the possible presence of re-
sidual monomer in these systems, M1 richest in AM
monomer and M6 richest in AA monomer that inde-
pendently of the exhaustive washings with double dis-
tilled water could have remained present.
4. Conclusions
Interpenetrated chitosan-poly(acrylic acid-co-acrylamide)
hydrogels with different compositions were synthesized
by the free radical polymerization of AM and AA in
presence of the CHI and MBA. Hydrogels swelling was
controlled by the interaction between the amino groups
of AM and the carboxylic groups of AA. Therefore, wa-
ter uptake decreased with increasing AA concentration in
the polymer and was also higher at pH 7.4. After treat-
ment with 0.1 N NaOH swelling capacity increased con-

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, 519
Characterization and Sustained Protein Release Studies
siderably due to AM hydrolysis but decreased considera-
bly with increasing ionic strength. Mechanical properties
of these systems can be tailored by adjusting hydrogel
composition i.e. for high mechanical strength hydrolysed
hydrogels prepared with 0.56 g AA, 0.16 g Am and 0.08
g CHI should be preferred. BSA loading capacity was
higher in (CHI/PAA/PAM)S hydrogels due to their high
water content and greater porosity.
Hydrogels were not cytotoxic for both systems except
for samples M1(QUI/PAA/PAM)A and M6(QUI/PAA/
PAM)A. BSA release experiments showed that these
hydrogels (hydrolysed and non hydrolysed) are capable
of sustained release at pH 6.8 and 7.4 after 10 h. BSA
released from (QUI/PAA/PAM)A and (QUI/PAA/PAM)S
hydrogels maintained its structural integrity at 6.7 and
7.4, which is a desirable requirement for preserving pro-
tein activity. The fact that (CHI/PAA/PAM)S hydrogels
exhibited limited BSA release in simulated gastric fluid
and sustained release in simulated intestinal fluids sug-
gests that they are good candidates as matrices for co-
lon-specific sustained protein release formulations.
5. Acknowledgements
M. Bocourt thanks the financial support for fellowship
program for doctoral training in the Cuba-Mexico Minis-
try for Foreign Affairs of Mexico. We like to thank Dr.
Ileana Echevarria Machado at the Biochemistry and Mo-
lecular Biology of Plants Department for UV spectros-
copy and Dr. Francis Avilés Cetina for mechanical test-
ing at the materials Department. This project is funding
from SEP-CONACYT. Project 2006-C01-61252, México.
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