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Calcium alginate/dextran methacrylate IPN beads as protectingcarriers for protein delivery
Giorgia D’Arrigo • Chiara Di Meo •
Laura Pescosolido • Tommasina Coviello •
Franco Alhaique • Pietro Matricardi
Received: 10 October 2011 / Accepted: 5 April 2012 / Published online: 24 April 2012
� Springer Science+Business Media, LLC 2012
Abstract In the present study, mechanical and protein
delivery properties of a system based on the interpenetra-
tion of calcium-alginate (Ca-Alg) and dextran-methacry-
late (Dex-MA) networks are shown. Interpenetrated
hydrogels beads were prepared by means of the alginate
chains crosslinking with calcium ions, followed by the
exposure to UV light that allows the Dex-MA network
formation. Optical microscope analysis showed an average
diameter of the IPN beads (Ca-Alg/Dex-MA) of 2 mm.
This dimension was smaller than that of Ca-Alg beads
because of the Dex-MA presence. Moreover, the strength
of the IPN beads, and of their corresponding hydrogels,
was influenced by the Dex-MA concentration and the
crosslinking time. Model proteins (BSA and HRP) were
successfully entrapped into the beads and released at a
controlled rate, modulated by changing the Dex-MA con-
centration. The enzymatic activity of HRP released from
the beads was maintained. These novel IPN beads have
great potential as protein delivery system.
1 Introduction
Controlled-release drug delivery systems are becoming
increasingly important in biomedicine, and a relevant
number of formulations have been developed. Potential
advantages of these systems include the maintenance of an
optimal therapeutic level, reduction of side effects by tar-
geting toward specific tissues, and improvement in patient
compliance by decreasing the frequency of administration
[1–4]. When therapeutic agents are proteins or peptides,
one of the major concern is the maintenance of their
activity during all the lifetime of the formulation, from the
preparation to the administration. Numerous protein and
peptide delivery systems have been widely investigated for
their ability to prevent the payload degradation [5–9].
Furthermore, formulations such as micro and nanoparticles,
[10–12] liposomes [13–15] and polymeric beads [16–19]
have been developed for a localized delivery of proteins
and peptides in a minimally invasive and sustained manner.
Hydrogels have been frequently proposed as potential
injectable carriers for modified drug delivery and tissue
engineering applications [20–22]. They maintain three-
dimensional structures with a high water content (up to one
thousand times their dry weight); moreover, their structural
similarity to the highly hydrated macromolecular-based
structures in the body, generally assures a good biocom-
patibility. Alginate, a natural biopolymer with a variety of
medical applications, can form hydrogels in mild condi-
tions in the presence of divalent cations such as Ca2?, Ba2?
and Sr2?, and was proposed as an injectable vehicle for
localized protein delivery [23]. Biocompatibility, low
toxicity, and low immunogenicity, both in vitro and in
vivo, [24, 25] encouraged the use of this polymer. Much
literature is devoted to the description of alginate micro-
particles, microcapsules and beads as drug delivery sys-
tems and as protein and enzyme carriers [26, 27]. The
modulation of the payload release is usually accomplished
by means of modification of the microparticle and bead
surfaces, in order to reduce the burst effects and increase
the microparticle stability in harsh environments. More
recently, a novel strategy based on polymeric blends has
been developed [28, 29]. In this case the synergistic effect
due to the presence of two polymers is exploited to prepare
G. D’Arrigo � C. Di Meo � L. Pescosolido � T. Coviello �F. Alhaique � P. Matricardi (&)
Department of Drug Chemistry and Technologies, ‘‘Sapienza’’
University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy
e-mail: [email protected]
123
J Mater Sci: Mater Med (2012) 23:1715–1722
DOI 10.1007/s10856-012-4644-0
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innovative drug delivery systems. Moreover, polymer
scaffolds or gels encapsulating particles for biomedical
applications have been reported. In this case the combi-
nation of polymeric microspheres in hydrogel macro-sys-
tems allows to combine mild preparative conditions for
protein entrapment (microspheres formation) and protein
controlled release (the hydrogel being the rate controlling
agent) [30–33]. In this context alginate microparticles
represent a very useful vehicle, due to the easy and mild
conditions needed for their preparation [34–36]. Within the
above mentioned contest, we developed new particulate
systems based on interpenetrating polymer network
hydrogels obtained with alginate and dextran methacrylate
derivative [36, 37]. Here the mechanical characterization
and the release properties of these new drug delivery sys-
tems are reported with respect to a reference system based
on plain alginate. In particular, it will be evidenced that the
novel IPN-based hydrogel shows, as main advantage, the
possibility to modulate the release of model proteins by an
appropriate tuning of its composition.
2 Materials and methods
2.1 Materials
Sodium alginate (Mw = 110 9 103, guluronic content:
65–75 %) was purchased from Carbomer; dextran (Mw =
40 9 103), glycidyl methacrylate (GMA) and 2,20-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were Fluka
products. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), 4-dymethylaminopyridine (DMAP), 2-hydroxy-40-(2-hydroxyethoxy)-2 methylpropiophenone (Irgacure 2959,
I2959) and Horseradish Peroxidase (HRP, MW 44 9 103, type
I) were from Sigma-Aldrich; bovine serum albumin (BSA,
MW 66 9 103), was an Acros Organics product. All other
chemicals were of reagent grade and were used without further
purification.
2.2 Synthesis of dextran methacrylate
Synthesis of dextran derivative was carried out according to
a modified procedure described in literature [38, 39]. In a
typical synthesis, 5 g of dextran and 1 g of DMAP were
solubilised in 50 mL of DMSO. Then, 2.2 mL of GMA was
added and the solution was kept in the dark for 48 h at 21 �C
under magnetic stirring. Finally, the reaction was stopped by
adding HCl 0.1 M until pH = 8 was reached. The solution
was exhaustively dialyzed (cut-off 14,000) against distilled
water, then freeze-dried, to obtain the dextran methacrylate
(Dex-MA) derivative. The degree of substitution (DS, i.e.,
the number of methacrylated groups per 100 glucopyranose
residues of dextran) was determined by 1H NMR in D2O
using a Bruker AC-400 spectrometer. A Dex-MA with
DS = 30 was prepared for the present work.
2.3 Preparation of Ca-Alg/Dex-MA beads
Aqueous solutions of sodium alginate (3 % w/V, pH
6.5–7.0) containing different concentrations of Dex-MA (5,
10, 15 or 20 % w/V) were prepared and aliquots (50 lL) of
I2959 solution (0.333 g in 1 mL of N-methylpyrrolidone)
were added. The polymeric solutions were then dropped
with the aid of a syringe (needle 22G) into a 0.5 M CaCl2solution and stirred for 5 min in order to cure the resulting
hydrogel. The obtained beads, recovered by filtration and
washed three times with 10 mL of distilled water, were
then irradiated by using an UV lamp (Perkin–Elmer,
Lambda 3A UV/Vis spectrophotometer, 1.213 mW) at
k = 365 nm for 10, 20 or 30 min in order to obtain beads
of calcium-alginate/dextran methacrylate (Ca-Alg/Dex-
MA). In Table 1 the various samples prepared are listed.
A3 is a calcium alginate sample prepared according to the
procedure above depicted without the addition of Dex-MA.
2.4 Beads dimension determination
The dimensions of the beads were evaluated using a Z16
APO Leica optical microscope, equipped with a Leica CLS
1509 halogen lamp support and a micrometer (sensitivity
50 lm). Aliquots of wet beads were analyzed. The appar-
ent radii of a population of 100 beads were estimated.
Reported data represent the mean values of three mea-
surements. All data are presented as the mean ± standard
deviation. Statistical analysis was performed with the
Student’s t test; a value of P \ 0.05 was considered as
statistically significant.
2.5 Hydrogel preparation for the rheological analysis
For the rheological analysis, hydrogels of Ca-Alg/Dex-MA
with the same composition and the same cross-linking time
applied for the formation of the beads (see Table 1) were
prepared. Briefly, 3 mL of 3 % w/V Alg-Na solutions (pH
adjusted to 6.5–7.0) containing different Dex-MA con-
centrations (5, 10, 15 or 20 % w/V) and 50 lL of I2959
solution (0.333 g in 1 mL of N-methylpyrrolidone) was
poured in petri dish, obtaining a solution layer B1 mm.
This polymer solution was covered with the same CaCl2solution used for the beads and the same curing time was
applied to simulate the diffusion of calcium ions into the
beads. After washing with distilled water, each sample was
then irradiated at k = 365 nm using an UV lamp (Perkin–
Elmer, Lambda 3A UV/Vis spectrophotometer, 1.213 mW)
for 10, 20 or 30 min, obtaining the Ca-Alg/Dex-MA
hydrogels.
1716 J Mater Sci: Mater Med (2012) 23:1715–1722
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2.6 Dynamomechanical and rheological analyses
The compression test on the Ca-Alg/Dex-MA beads was
performed by means of a Texture Analyzer TA-XT2i (Stable
Micro Systems, UK), with a 5 kg load cell, using a 5 mm-
diameter stainless steel cylindrical probe. Each bead was
analyzed at room temperature using a trigger force of 0.02 N
and a strain in compression of 80 %, repeating the measure
for 10 beads for each type of sample. The force needed for the
80 % deformation of the sample was recorded as Fmax. The
corresponding cohesion energy, Ecohes, was determined as
the positive area under the force—time curve from zero to
the Fmax [40]. Rheological analysis was carried out on Ca-
Alg/Dex-MA hydrogels having the same compositions of the
beads, a diameter of 5 cm and a height of 0.5–0.8 mm,
prepared as described (Sect. 2.5) above. The analyses were
performed using a controlled stress Haake RheoStress 300
Rotational Rheometer, equipped with a Haake DC10 ther-
mostat; oscillatory experiments on the hydrogels were car-
ried out at 25 �C in the range 0.01–50 Hz using a grained
plate–plate device (Haake PP35 TI: diameter = 35 mm),
performing preliminary stress sweep experiments
(f = 1 Hz, s = 0.001–1,000 Pa, T = 25 �C) to assess the
linear viscoelastic response range of the samples. The used
deformation c was always lower than 0.005.
In order to calculate the crosslinking density for each
IPN system, the equation: Cd = G/RT, where G is the
shear modulus calculated as the mean G’ value in the
frequency range studied, R is the universal gas constant and
T the absolute temperature, was exploited [41].
2.7 In vitro release studies
For the BSA release studies, the beads were prepared as above
described, adding 6 mg of BSA to the initial Alg/Dex-MA
solutions; the UV cross-linking time was fixed at 10 min.
The BSA loaded beads were suspended at 37 �C in
200 mL of HEPES buffer (1 mM, pH = 7.4 by addition of
NaOH 1 N) for 24 h. Then, the beads were treated with
EDTA solution (2 % w/V) to dissolve the Ca-Alg hydrogel
and to obtain the release of the protein still entrapped
within the matrix in order to evaluate the total amount
loaded by the beads.
Released protein was spectrophotometrically quantified
using the Bradford microassay colorimetric method, by
mixing for 5 min 1.5 mL of the samples and 1.5 mL of
Bradford reagent brilliant blue G and recording the
resulting absorbance at k = 595 nm [42]. The protein
amount in the samples was determined by a calibration
curve obtained in the same experimental conditions. In
order to simulate gastric conditions, the BSA loaded beads
(Sect. 2.3), were suspended at 37 �C in 200 mL of HCl
0.1 N (simulated gastric fluid) for 2 h, then in HEPES
buffer (1 mM, pH = 7.4 by addition of NaOH 1 N) for the
following 22 h. Then, the beads were treated with EDTA
solution (2 % w/V) to dissolve the Ca-Alg gel and to obtain
the release of the protein still entrapped within the matrix
in order to evaluate the total amount loaded by the beads.
The HRP enzyme was loaded in the Ca-Alg/Dex-MA
beads following the same procedure, and the release was
studied in HEPES at 37 �C for 24 h. HRP quantification was
performed by Bradford Microassay as above reported for
BSA; the HRP activity was evaluated using the redox system
ABTS/H2O2: for this purpose, 30 lL of the sample con-
taining HRP and 30 lL of H2O2 1 mM were added to 3 mL
of ABTS solution 77.7 lM at 25 �C. The formation of rad-
ical monocation ABTS? was recorded at k = 414 nm, at the
3 and at the 4 min, to evaluate the activity of the released
enzyme. The activity of the released enzyme was tested
measuring the oxidation rate of ABTS [43, 44]. The enzy-
matic efficiency was calculated according to:
Ast : Cst = A� : C,
where Ast is the absorbance value of the standard solution
at concentration Cst, C is the concentration of the tested
solution, obtained by the Bradford method, and A* is its
assumed absorbance. The enzymatic efficiency (EE) is then
calculated as:
A414
A�= % EE
where A414 is the absorbance of the tested solution recor-
ded at 414 nm.
Table 1 Beads prepared with different polymer compositions,
concentrations and crosslinking times
Sample [Ca-Alg]
(% w/V)
[Dex-MA]
(% w/V)
Time of cross-
linking (min)
A3 3 – –
A3D530 3 5 –
A3D530t10 3 5 10
A3D530t20 3 5 20
A3D530t30 3 5 30
A3D1030 3 10 –
A3D1030t10 3 10 10
A3D1030t20 3 10 20
A3D1030t30 3 10 30
A3D1530 3 15 –
A3D1530t10 3 15 10
A3D1530t20 3 15 20
A3D1530t30 3 15 30
A3D2030 3 20 –
A3D2030t10 3 20 10
A3D2030t20 3 20 20
A3D2030t30 3 20 30
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3 Results and discussion
3.1 Beads characterization
3.1.1 Beads size evaluation
Ca-Alg/Dex-MA beads prepared by applying different
cross-linking times were observed with the optical micro-
scope in order to evaluate their average diameter; the
results are reported in Fig. 1. Obtained data show a
decrease of the average diameter of the beads as Dex-MA
concentration increases; moreover the cross-linking reac-
tion leads to a reduction of the bead dimensions, most
probably due to a contraction of the structure related to the
formation of the chemical network.
3.1.2 Mechanical properties
In order to investigate the effect of polymer concentration
and crosslinking conditions on the mechanical properties of
the beads, mechanical tests in large deformation conditions
were performed. Data of compression tests carried out on
beads at different compositions and crosslinked for dif-
ferent crosslinking times are reported in Table 2. As an
example, experimental curves related to the A3D2030txsamples are reported in Fig. 2.
0
10
20
30
40
50
60
70
d (mm)
30 min 20 min 10 min 0 min
05
1015202530354045
d (mm)
30 min 20 min 10 min 0 min
05
1015202530354045
d (mm)
30 min 20 min 10 min 0 min
% n
um
ber
% n
um
ber
% n
um
ber
1.8 1.9 20 2.1 2.2 2.3 2.4 2.5 2.6 2.7
1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6
1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7
0
10
20
30
40
50
60
d (mm)
30 min 20 min 10 min 0 min
% n
um
ber
1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5
Bead samplesAverage Diameter
(mm ± σ)
A3 2.7 ± 0.1
A3D530
A3D530t10 2.1 ± 0.1
A3D530t20 2.1 ± 0.2
A3D530t30 2.2 ± 0.1
A3D1030 2.4 ± 0.1
A3D1030t10 2.2 ± 0.2
A3D1030t20 2.2± 0.2
A3D1030t30 2.1± 0.1
A3D1530
A3D1530t10 2.0 ± 0.2
A3D1530t20 2.0 ± 0.1
A3D1530t30 2.0 ± 0.1
A3D2030
A3D2030t10 2.1 ± 0.1
A3D2030t20 2.0 ± 0.1
A3D2030t30 2.0± 0.1
a
b
c
d
2.5 ± 0.1
2.4 ± 0.1
2.3 ± 0.2
Fig. 1 Mean diameters of
Ca-Alg/Dex-MA beads
prepared using Dex-MA
(DS 30 %) at different
crosslinking times and different
concentrations: a 5 % (w/V),
b 10 % (w/V), c 15 % (w/V)
and d 20 % (w/V)
1718 J Mater Sci: Mater Med (2012) 23:1715–1722
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For not cross-linked beads a decrease in Fmax is
observed as Dex-MA concentration increase, because of
the possible interference of Dex-MA chains on Ca-Alg
‘‘egg boxes’’, [45, 46] that leads to a reduction of the
polymer network strength. On the other side, for the cross-
linked beads, both the Fmax and the Ecohes increase as Dex-
MA concentration in the beads increases. Fmax and Ecohes
are only slightly dependent on the crosslinking time. This
can be ascribed to the low sensitivity of the dynamome-
chanical equipment in large deformation experiments.
3.1.3 Rheological analysis
Rheological analyses in oscillatory shear experiments on
Ca-Alg/Dex-MA hydrogels were carried out to investigate
in linear regime (small deformation conditions) the effect
of composition and crosslinking time on the obtained net-
work. In this case, being impossible to study beads in shear
conditions, hydrogels with an analogous composition were
prepared in a thin layer shape (lesser than 1 mm), as
described in the experimental part.
Obtained data showed a strong-gel behavior for all
systems tested. In Fig. 3 the mechanical spectra for the
A3D530tx gel systems are reported as an example.
In Fig. 4a, b the elastic moduli G’, recorded at 1 Hz, as
a function of Dex-MA concentration and crosslinking time
are respectively reported. It can be easily observed that G’
increases as the Dex-MA concentration increases, showing
the same trend observed for the Fmax data obtained in large
deformation experiments. Moreover, the crosslinking time
influences the elasticity of the resulting hydrogels; in par-
ticular it can be observed a remarkable effect for the sys-
tems with the higher concentration of Dex-MA, i.e. 15 %;
actually, the increase is more than six times after 30 min
crosslinking with respect the system not irradiated. For the
other samples, 5 and 10 % Dex-MA, a doubling of G’ was
registered in the same time.
Rheological characterisation allows an estimation of the
polymer network nanostructure via the determination of the
cross-link density, Cd, defined as the moles of junctions
between different polymeric chains per hydrogel unit vol-
ume [41]. Obtained data, reported in Table 2, clearly
indicate that the systems prepared have an high crosslink-
ing density and that this density markedly increases as the
methacrylate content increases. For the samples not UV
crosslinked, this density, on the contrary, decreases, sup-
porting the hypothesis of the disturbing effect of methac-
rylate moieties on the ‘‘egg box’’ formation between Ca2?
and alginate chains [45, 46].
Table 2 Maximum compression forces, cohesion energies and
crosslinking densities calculated for Ca-Alg/Dex-MA beads at dif-
ferent composition and crosslinking time
Sample Fmax (N) ± (r) Ecohesion (mJ) ± (r) Cd (mol/m3)
A3 1.04 ± 0.05 0.79 ± 0.08
A3D530 0.92 ± 0.10 0.64 ± 0.06 7.7
A3D1030 0.84 ± 0.10 0.44 ± 0.05 4.0
A3D1530 0.64 ± 0.05 0.35 ± 0.05 7.5
A3D2030 0.79 ± 0.09 0.53 ± 0.05 3.3
A3D530t10 1.00 ± 0.03 0.70 ± 0.03 9.5
A3D1030t10 1.02 ± 0.10 0.80 ± 0.03 11.3
A3D1530t10 1.22 ± 0.07 0.82 ± 0.12 18.0
A3D2030t10 1.35 ± 0.07 0.94 ± 0.002 22.4
A3D530t20 1.00 ± 0.03 0.71 ± 0.05 11.6
A3D1030t20 1.02 ± 0.13 0.82 ± 0.01 14.6
A3D1530t20 1.23 ± 0.22 0.86 ± 0.12 24.2
A3D2030t20 1.34 ± 0.18 0.92 ± 0.10 27.2
A3D530t30 1.10 ± 0.13 0.74 ± 0.08 11.2
A3D1030t30 1.12 ± 0.08 0.82 ± 0.08 13.3
A3D1530t30 1.27 ± 0.11 0.89 ± 0.13 28.2
A3D2030t30 1.40 ± 0.09 1.05 ± 0.10 48.8
40
Fo
rce
(N)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 20 60 80 100
strain (%)
A3D2030A3D2030t10
A3D2030t20
A3D2030t30
Fig. 2 Compression test profiles of A3D2030tx beads crosslinked for
10, 20 and 30 min
100
1000
10000
100000
0.01 0.1 1 10 100
f(Hz)
G'(P
a), G
''(P
a)
G' A3D530t10a G'' algret 10 min
G' alg320min G'' alg3%et 20 min
G' alg3%ret 30 min G'' alg3t 30 min
G' alg non ret
G A3D530t10
G A3D530t20
G A3D530t30
G A3D530
G A3D530t10
G A3D530t20
G A3D530t30
G A3D530
Fig. 3 Mechanical spectra of A3D530tx hydrogels
J Mater Sci: Mater Med (2012) 23:1715–1722 1719
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3.2 Protein release
The protein loading procedure adopted in the present work
is extremely easy to perform but its efficiency is not
complete. During the bead curing in the gelling Ca2?
solution, the protein can diffuse out of the beads thus
lowering the payload. In developing experimental set up, a
balance was thus stricken between the curing time (influ-
encing the mechanical properties of the beads) and loosing
the payload. It is worth noting that also the Ca2? concen-
tration of the solution is an important parameter as well, as
it can influence Ca2? diffusion into the beads, thus
affecting the ‘‘egg box’’ formation and, consequently, the
mechanical and drug delivery properties of the obtained
systems. [47] According to the beads preparation procedure
in the present work, the entrapment efficiency of BSA by
the Ca-Alg/Dex-MA beads prepared at different composi-
tions was about 60 % for all tested systems.
After the entrapment, the capability of the beads to
modulate the BSA release in various conditions was
also tested. For this purpose, A3D530t10, A3D1030t10,
A3D1530t10 and A3D2030t10 beads were chosen as drug
delivery systems, in order to expose the loaded protein to
the lowest UV dose; A3 beads were used for an appropriate
comparison.
In neutral conditions (pH 7.4, HEPES 1 mM) the beads
A3D530t10 and A3D1030t10, characterized by the lower
Dex-MA content, showed a complete protein release within
24 h, similar to that of A3. The samples A3D1530t10 and
A3D2030t10, with higher methacrylate moieties content, as
well as higher polymer content, showed a not complete
release even after 24 h, as shown in Fig. 5. Further
experiments were carried out to assess the ability of the
prepared beads to limit the protein diffusion in acidic
conditions, thus acting as pH sensitive delivery systems.
The release profiles, reported in Fig. 6, indicate that in HCl
medium (simulated gastric fluid) all samples showed only
an initial burst effect, that can be ascribed to the release of
BSA present on the outer shell of the beads, while a further
release of the protein is inhibited due to the alginic acid
hydrogel formation and to the subsequent reduction of the
porosity of the system. When the solvent was changed to
HEPES medium pH 7.2, the beads start to release the BSA
molecules. The samples A3D530t10 and A3D1030t10 show a
release profile very similar to that of the A3 beads, with an
almost complete protein release within 24 h. Again,
A3D1530t10 and A3D2030t10 beads show a slower BSA
00 5 10 15 20 25 30 35
cross-linking time (min)
Dex-MA% (w/V)
00 5 10 15 20
10 min 20 min 30 min 0 min10 min 20 min 30 min 0 min10 min 20 min 30 min 0 min
A3D530tx
A3D1030tx
A3D1530tx
A3D2030tx
10 min 20 min 30 min 0 min10 min 20 min 30 min 0 min
20
40
60
80
100
120
140
20
40
60
80
100
120
G'(
kPa)
at
1 H
zG
'(kP
a) a
t 1
Hz
140a
b
Fig. 4 Elastic modulus G’ measured at 1 Hz as function of:
a Dex-MA concentration (% w/V) and b cross-linking time
0
20
40
60
80
100
0 4 8 12 16 20 24
time (h)
% B
SA
rel
ease
d
A3
A3D530t10
A3D1030t10
A3D1530t10
A3D2030t10
Fig. 5 Release profiles of BSA from Ca-Alg/Dex-MA beads at
different compositions in HEPES 1 mM, pH 7.4, T = 37 �C. Results
obtained for beads of alginate (A3) are also reported for comparison
0
20
40
60
80
100
0 4 8 12 16 20 24
time (h)
% B
SA
rel
ease
d
A3
A3D530t10
A3D1030t10
A3D1530t10
A3D2030t10
Fig. 6 Release profiles of BSA from Ca-Alg/Dex-MA beads at
different compositions in HCl 0.1 M for 2 h, then in HEPES 1 mM
pH 7.4, T = 37 �C. Results obtained for beads of alginate (A3) are
also reported for comparison
1720 J Mater Sci: Mater Med (2012) 23:1715–1722
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release, as no more than 60 % of the total amount of loaded
protein is released after 24 h.
As a general behavior, it seems that IPN beads with an
high methacrylate content can entrap in a not reversible
manner about 40 % of the loaded BSA; it can be argued
that hydrophobic interaction as well as physical entrapment
in the beads core can be related to a some non homoge-
neous distribution of the polymer chains within the IPN
system. It can be supposed that during the hydrogel curing
by means of Ca2?, a segregation of the Alg chains
(involved in the ‘‘egg box’’ formation) from the Dex-MA
chains can occur, thus inducing the formation in the centre
of the beads (due to the spherical symmetry of the beads,
Ca2? diffuses from the surface of the polymer solution
drop toward the centre) of a core rich in methacrylate
content. After the UV curing, the polymer network in this
region can be assumed to be tighter than in the surrounding
thus limiting the diffusion of the loaded molecules that
result almost entrapped. This effect is, obviously, polymer
concentration dependent. To test whether the beads prep-
aration can modify the protein structure, HRP (isoelectric
point pH 7.2), chosen as a model enzyme, was loaded in
the IPN beads. The release of HRP from the beads was
evaluated in physiological conditions. The entrapment
efficiency of HRP by the Ca-Alg/Dex-MA beads was
always between 70 and 75 %.
In Fig. 7a HRP delivery profiles are reported. The release
occurs quite rapidly with respect to that of BSA for all bead
systems: according to the lower MW of HRP than that of
BSA, the enzyme can diffuse out of the beads more easily and
more than 80 % of the entrapped protein is released in 24 h.
In particular, it has been observed that also the beads with the
higher Dex-MA content, release after one hour more than
80 % of the entrapped protein. The results, reported in
Fig. 7b, show that the enzyme activity is preserved for the
first 8 h, while a slight decrease was observed after 24 h,
clearly indicating that the beads preparation conditions are
mild, thus preserving protein activity.
4 Conclusions
The present work describes the formation and the charac-
terization of beads based on Ca-Alg/Dex-MA hydrogels.
The interpenetration of these polysaccharides and their
networks in the beads influenced the physico-chemical
properties as well as the protein release of these particulate
systems. The beads dimensions decreased with the pres-
ence of Dex-MA in the formulation and it was more evi-
dent after the formation of the Dex-MA chemical network.
Also the strength of the beads, and of their analogous
hydrogels, was related to the Dex-MA concentration and to
its crosslinking time which influenced, in particular, the
elasticity of the systems. The Ca-Alg/Dex-MA beads
showed a good efficiency in entrapment of model proteins
(60 and 75 % for the BSA and the HRP, respectively) and
the possibility to tune the release of these proteins varying
the Dex-MA concentration in the beads. More importantly,
the studies of the activity of the HRP delivered from the
beads showed the preservation of the enzyme activity
during the release test. Obtained data indicate that IPN
beads can represent an useful tool for the administration of
therapeutic proteins as mechanical and delivery properties
can be easily and appropriately tuned by modifying the IPN
composition.
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