European Polymer Journal 52 (2014) 137145

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European Polymer Journal

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

Macromolecular Nanotechnolgy

A novel method for hydrogel nanostructuring

Ortal Yom-Tov a, Ilya Frisman b, Dror Seliktar c,d, Havazelet Bianco-Peled b,d,
a

Inter-Departmental Program for Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel
Department of Chemical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel
c
Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel
d
The Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa 32000, Israel
b

i n f o

Article history:
Received 25 August 2013
Received in revised form 17 December 2013
Accepted 5 January 2014
Available online 11 January 2014
Keywords:
Hydrogel
Structure
SAXS
Nanostructuring

a b s t r a c t
Nanostructured hydrogels tailor-made for specic applications are new grounds for the
creation of novel soft materials. The current research presents a new method for hydrogel
nanostructuring involving the incorporation of Pluronic F127 micelles mixed with acrylated blockcopolymer molecules, which enable the attachment of these micelles to the
hydrogel matrix through their endgroups. This design impacts the hydrogel nanostructure
as well as its swelling and mechanical properties. Small Angle X-ray Scattering (SAXS) and
Cryogenic Transmission Electron Microscopy (cryo-TEM) revealed that photochemical
crosslinking of the hydrogel caused immobilization of the nanostructured micelles.
Mechanical and weight gain experiments demonstrated a signicant impact of these nanostructures on the hydrogels elastic modulus as well as the transient and equilibrium
weight gain ability of the material.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Nanostructuring is becoming increasingly important in
the design of precisely dened 3D structures which enable
control over the material characteristics. In principle, by
optimizing the nanostructure, materials tailor-made for a
specic application can be created with structural denition to a sub-cellular level. The development of hydrogel
nanostructuring methods are still challenging because
the high water content excludes the use of lithographic
techniques. One approach for nanostructuring hydrogels
utilizes the self-assembly capability of block and graft
copolymers driven by hydrophobic interactions between
the blocks [13]. For example, nanostructured poly(e-caprolactone)-poly(ethylene
oxide)-poly(e-caprolactone)
(PCL-b-PEO-b-PCL) hydrogels were created by PCL removal
after synthesis in order to create a nanoporous structure
[4,5]. The incorporation of nanometric scaled structures
Corresponding author at: Department of Chemical Engineering,
Technion-Israel Institute of Technology, Haifa 32000, Israel. Tel.: +972 4
8293588; fax: +972 4 8295672.
E-mail address: bianco@tx.technion.ac.il (H. Bianco-Peled).
0014-3057/$ - see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.eurpolymj.2014.01.004

with different morphologies into the hydrogels contributed to the control over the hydrogel nanostructure,
mechanical and physical properties [68]. Poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels incorporated with
core cross-linked poly(ethylene glycol)-block-poly(e-caprolactone) (PEG-b-PCL) micelles having spherical or rodlike morphologies were likewise prepared and evaluated
for use as drug-eluting soft contact lenses [6]. Integration
of micelles with crosslinked cores into pHEMA hydrogels
led to the formation of different internal nanostructures
which were dependent on the amount and morphology
of the embedded micelles. Incorporation of cross-linkable
micelles was found to reduce the degree of hydrogel
weight gain. Combining hexagonal or lamellar lyotropic liquid crystals with poly(ethylene glycol) diacrylate (PEGDA) hydrogels was found to impact their physical
properties including network swelling, mechanics, and
degradation [7]. Nanostructuring of hydrogels during their
formation has recently been reported by our group [8]. This
approach utilizes the self-assembly ability of biocompatible, amphiphilic block-copolymers of poly(ethylene
oxide)/poly(propylene oxide) (Pluronic) in order to

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a r t i c l e

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impart nanostructured organization in the hydrogels during photopolymerization. Limited stability of these micelles was observed, mostly attributed to their diffusion
out of the hydrogels after four days in aqueous buffer. In
fact, the gradual escape of Pluronic molecules from the
hydrogel resulted in a signicant change to the nanostructure of the polymer network.
The main objective of the current study was to design a
novel method for nanostructuring of hydrogels. This method is based on embedding Pluronic micelles in a hydrogel
while anchoring some of their molecules to the surrounding network through their endgroups. Our hypothesis was
that in this design, the anchored molecules could provide a
means to further crosslink the hydrogel and stabilize the
network. In contrast to traditional low molecular weight
crosslinkers, the covalently bound molecule can stretch
as the unbound Pluronic diffuse out of the hydrogel and
the micellar structures are lost. We postulated that this unique design will allow further manipulation of the gel
properties. Herein we describe a comprehensive analysis
of hydrogels prepared using this new nanostructuring
methodology. Small angle X-ray scattering (SAXS) and
transmission electron microscopy at cryogenic temperature (cryo-TEM) were used for structural characterization,
whereas mechanical and weight gain experiments were
used to explore the impact of nanostructure alterations
on these properties.
2. Experimental
2.1. Synthesis of PEG diacrylate (PEG-DA) and F127 diacrylate
(F127-DA)
F127-DA and PEG-DA were prepared from Pluronic
F127 (BASF) and poly(ethylene glycol) (PEG, molecular
weight 10 kDa), respectively, as described elsewhere [9].
Briey, acrylation was carried out under Argon by reacting
the polymers with Acryloyl-chloride (Merck, Darmstadt,
Germany) and triethylamine (TEA) (Fluka) at a molar ratio
of 150% relative to the hydroxyl groups. The resulting
product was precipitated and dried under vacuum for 48 h.
Preparation of PEG-brinogen (PF) precursor solution is
described elsewhere [8]. Briey, bovine plasma brinogen
(SigmaAldrich) was dissolved in phosphate buffer saline
(PBS) containing 8 M Urea. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (SigmaAldrich) was added
to completely dissolve the protein dissolution, the pH
was adjusted to 8, and PEG-DA (10 kDa) in PBS solution
was added while maintaining a ratio of 4:1 PEG-DA to protein cysteines. After the reaction was completed, the product was diluted, precipitated, redissolved, homogenized
and dialyzed against 150 mM PBS at 4 C for 24 h with
two changes of PBS (Spectrum, 1214-kDa MW cutoff, California, USA). Protein concentration was determined by
Bis-Cinchoninic Acid (BCA) protein assay.
2.2. Nanostructured PF hydrogels
Mixtures of Pluronic F127 and F127-DA at different ratios were prepared with PF precursor solution (protein

concentration 8.5 0.5 mg/ml) at 4 C until complete dissolution of the block-coloymers was achieved. Total concentration of the block-copolymer (Pluronic F127 plus
F127-DA) was kept constant at 10% (w/v). The solution
was mixed with 0.1% (v/v) photoinitiator stock solution
containing 10% (w/v) Irgacure2959 in 70% ethanol and
30% deionized water. The hydrogel precursor solution
was heated to 37 C for 10 min in order to induce micelle
formation, followed by irradiation with UV light (365 nm,
45 mW/m2) for 5 min in order to achieve a chemically
crosslinked hydrogel.
Small Angle X-ray Scattering (SAXS) experiment were
performed using a small-angle diffractometer (Molecular
Metrology SAXS system) with Cu Ka radiation from a
sealed microfocus tube (MicroMax-002+S), two Gbel mirrors, and three-pinhole slits (generator powered at 45 kV
and 0.9 mA). The scattering patterns were recorded by a
20  20 cm two-dimensional position sensitive wire detector (gas lled proportional type of Gabriel design with
200 lm resolution) that was positioned 150 cm behind
the sample. The resolution of the SAXS system was approximately 23 nm1. The scattered intensity I(q) was recorded in the interval 0.07 < q < 2.7 nm1, where q is the
scattering vector dened as q = (4p/k)sin(h), 2h is the scattering angle, and k is the radiation wavelength
(0.1542 nm). The sample under study was sealed in a
thin-walled glass capillary of about 2 mm diameter and
0.01 mm wall thickness, and measured under vacuum at
constant temperature. The I(q) was normalized to the following parameters: time, solid angle, primary beam intensity, capillary diameter, transmission, and the Thompson
factor. Scattering from the solvent, empty capillary and
electronic noise were subtracted. SAXS curves were measured at q vs. I, where I has the units of (1/nm3).
Cryogenic Transmission Electron Microscopy (cryo-TEM)
micrographs were obtained from ultra-fast cooled vitried
cryo-TEM specimen prepared under controlled conditions
37 C and 100% relative humidity as described elsewhere
[10]. Specimens were examined in a Philips CM120 cryoTEM operating at 120 kV, using an Oxford CT3500 cooling-holder system that kept the specimens at about
180 C. Low electron-dose imaging was performed with
a Gatan Multiscan 791 CCD camera, using the Gatan Digital
Micrograph 3.1 software package.
Samples for water weight gain experiments were prepared by using round Teon molds with diameter of
14 mm, whereby PF hydrogels with the addition of Pluronic F127/F127-DA mixtures at different ratios were tested.
The control groups included PF hydrogels with the addition
of PEG-DA at different percentages. Each sample was made
by transferring precursor solution (0.5 ml) into the Teon
mold, heating the solution to 37 C for 10 min and then
crosslinking the hydrogel with UV light for 5 min as described before. The hydrogels were subsequently submerged in 150 mM PBS containing 0.2% sodium azide in
Petri dishes. The dishes were incubated at 37 C and the
water weight gain ratio was determined gravimetrically
as follows:

%Weight gain

mW  mD
 100
mW

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O. Yom-Tov et al. / European Polymer Journal 52 (2014) 137145

Mechanical characterization was performed using a

Lloyd Tensile machine in compression mode. The sample
preparation protocol, compositions and size were identical
to the ones used in the swelling experiments. The
samples were compressed at a rate of 1 mm/min. The
Youngs modulus was calculated from the slope between
1% and 10% extensions in the linear region of the stress
strain curve [11]. The average values and standard deviations were obtained from the analysis of at least 8
measurements.

The current research explores the relationship between

architecture and properties using immobilized Pluronic
F127 micelles in PEG-brinogen hydrogels. Covalent linkage of the micelles into the matrix was achieved by replacing part of the Pluronic F127 molecules with an acrylated
derivative, F127-DA, which can react with the PF precursor
molecule during the free-radical polymerization reaction
that leads to hydrogel formation. In order to isolate the
inuence of Pluronic F127 acrylation from other parameters, such as micelle size and density, the overall blockcopolymer concentration was kept constant at 10% (w/v).
Therefore, the main variable in the experimental design
of this study was the ratio between Pluronic F127 and
F127-DA. This experimental setup allowed for variations
in the amount of molecules bound to the matrix and consequently, the alteration of stable nanostructural features
that resulted in the hydrogels, including mesh size and
micellar arrangement.

3.1. Precursor solutions with added Pluronic F127/F127-DA

mixtures

Fig. 1. (A) Schematic representation of aggregated PF. (B) Schematic

representation of Pluronic F127 micelles. (C) Normalized scattering
intensity curves of PF hydrogels with (s) 2.5% F127-DA and 7.5%
Pluronic F127, (D) 5% F127-DA and 5% Pluronic F127, (e) 7.5% F127DA and 2.5% Pluronic F127 and (h) 10% F127-DA. The intensity curves
were multiplied by a factor for better visualization. Fits to Eq. (11),
calculated from the best-t parameters summarized in Table 1, are shown
as solid lines.

equation describing the scattering as a cylinder with

length L and radius of gyration Rg [14]:

Icyl K cyl L

p
q

 e

q2 R2
g

Precursor solutions containing Pluronic F127/F127DA at different ratios were investigated in order to observe if the block-copolymer mixtures self-assemble at
37 C in a similar fashion to solutions of precursor with
10% (w/v) unreactive Pluronic F127. The structure of
the three-component PF/Pluronic F127/F127-DA system
is rather complex, nevertheless the structure of each of
the individual components was previously subject to a
substantial analysis. Our previous SAXS and SANS analysis
of PF precursor showed that these molecules aggregated
in solution due to proteinprotein interactions, and
formed elongated cylinder-like objects with grafted PEG
chains [12], as schematically illustrated in Fig. 1A. This
aggregation is likely owing to the nature of the PF
precursor, a semi-synthetic molecule composed of a
hydrophobic Fibrinogen backbone with grafted hydrophilic PEG chains [13]. The grafted chains on the PF carry
saturated endgroups that allow network formation via
photopolymerization reaction.
Our previous study [12] demonstrated that SAXS curves
only reect the contribution of the brinogen backbone
and not that of the attached PEG chains because the electron density of PEG (3.45  107 3) is almost identical to
that of the solvent (water, 3.34  107 3). The scattering
patterns of the PF solution alone was thus tted to an

where Kcyl is a constant.

Pluronic F127 at concentration of 10% (w/v) was previously shown to form closely packed micelles [1517]. Because the corona of these micelles consisted of PEG
chains with low contrast, the scattering from the micelles
Imicelle is calculated by multiplying the form factor P(qR),
which describes the shape of a spherical core with radius
R, and the structure factor S(qRHS) which describes particle
interference with interaction radius of RHS between neighboring hard spheres. The PercusYevick approximation for
hard spheres was used to calculate the structure factor
[18]. Taking both the form factor and the structure factor
into account, the following equations were used to describe scattering from Pluronic F127 micelles, shown
schematically in Fig. 1B [18]:

Imicelle u 

4p 3
R  Dq2  PqR  SqRHS
3

where u is the volume fraction of the cores and Dq is the

electron density difference between the core and the medium. The shape factor is given by [18]

"
PqR 3

sinqR  qR cosqR
qR3

#2
4

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3. Results and discussion

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The structure factor takes the form:

1
1 24hb GA=A

SqRHS

in the micrographs of these materials, as can be seen in

Fig. 2.

where hb is the volume fraction of the hard spheres and

A 2qRHS
GA a

2A sinA 2  A2 cosA  2
b
A2
A3
4
2
fA cosA 43A  6 cosA A3  6AsinA 6g

sinA  A cosA

A5
7

1 2hb 2

MACROMOLECULAR NANOTECHNOLOGY

b 6hb

1  hb 4
1 hb =22

3.2. Nanostructured hydrogels with added Pluronic F127/

F127-DA mixtures
Nanostructured PF hydrogels containing Pluronic
F127/F127-DA at various ratios were studied with the purpose of determining whether the attachment of micelles to
the hydrogel alters the nanostructures. SAXS patterns of
the nanostructured hydrogels are shown in Fig. 3A.
Increasing the relative percentage of F127-DA resulted in
a smearing and a shifting of the inter micellar peak to
smaller scattering angles. This outcome hinted to an
alteration in the nanostructure of the hydrogels (after
crosslinking) which could be ascribed to the binding of
the micelles onto the PF network. With an increase in the

1  hb 2

ha
2

10

Finally, the scattering from the PF solution containing

Pluronic F127/F127-DA is described by summing Eqs.
(2) and (3):

Iq Icyl Imicelle
K cyl L

p
q

e

q2 R2
g
2

u

4p 3
R  Dq2  PqR  SqRHS
3

11

A good t of Eq. (11) to the experimental SAXS data

(Fig. 1C) conrmed that micelles are formed in solution
at 37 C, as initially assumed. Close packing of the micelles
is evident from the structural peak observed at small scattering angles. As can be expected from the similarities between the scattering patterns, there were no
distinguishable differences between the calculated parameters of the different solution samples, which indicated
that altering the ratio between Pluronic F127 and F127DA has no remarkable inuence on the nanostructure.
The tted core radius is about 80 , in agreement with previously reported values for Pluronic F127 in water at
37 C [10].
Cryo-TEM was used to directly observe micelle formation in solutions of PF containing F127. Because the contrast between Pluronic F127 molecules to the
background was very low, increased exposure time of the
irradiated electron beam was applied to the tested samples. This technique was previously used in cryo-TEM studies of Pluronic micelles to enhance contrast [10]. In order
to ensure that the nanostructures in the sample were not
as a result of damage caused to the sample by overexposure to radiation, short exposure times were also used in
control measurements. The selective electron beam etching exposed micellar structures in the PF samples containing Pluronic F127. Because the PF solutions containing the
different percentages of F127-DA demonstrated similar
scattering patterns, only PF solution with 10% w/v added
F127-DA was studied by Cryo-TEM as a representative
example. A well-ordered micelles structure was observed

Fig. 2. Cryo-TEM images of PF solution with 10% F127-DA at magnication of (A) X66 and (B) X175.

When tting the experimental SAXS data of the PF and

F127-DA materials to Eq. (11), we found that the model
which does not account for any variations in the cylinders
dimensions did not result in as good a t to this data when
compared to the t for PF materials alone. In contrast, a
similar model with variations in the cylinders dimensions
resulted in a much better t of the experimental SAXS data
for the PF and F127-DA materials. Therefore, it was assumed that the PF protein size variations are related to
the increasing concentrations of F127-DA in the precursor
solution [12]. This could be attributed to the aggregation of
the protein backbone occurred during crosslinking; consequently two populations of proteins with different sizes
are now present: one population preserves the original radius of approximately 2 nm and a second population
whose size varies as a function of crosslinker type and concentration. For simplicity, the revised model approximated
the cylinder size distribution using two populations of cylinders with different radii, and Eq. (11) was adjusted
accordingly. The best t parameters to the revised Eq.
(11) are summarized in Table 1, and supported the qualitative observations made based on the shape of the curves.
Increasing the relative percentage of F127-DA interferes
with the ability of the Pluronic F127 molecules to selfassemble into spherical micelles. As a result, the distance
between neighboring existing micelles RHS increased and
their volume fraction hb decreased. Comparing the t
parameters to those obtained from the corresponding solutions showed that the size of the protein backbone was
also affected by the chemical crosslinking reaction (i.e.
photopolymerization). Specically, the fact that two different sizes of cylinders with different radii had to be used to
achieve a better t to the experimental SAXS data underscores the observations regarding F127-DA. Moreover, larger aggregates were evidently formed when more F127-DA
was present. Thus, enhancing the micelle stability by
immobilizing them in the PF network resulted in further
aggregation of the protein backbone and the formation of
larger, more size dispersed aggregates. As stated above,
additional changes to the network due to the increased
percentage of micelles bound to the PF network include
larger distances between the micelles and reduced degree
of ordering. These main ndings are summarized in the

Fig. 3. (A) Normalized scattering intensity curves of PF hydrogels

containing 10% block copolymer at various Pluronic F127/F127-DA
ratios (s) 2.5% F127-DA and 7.5% Pluronic F127, (D) 5% F127-DA and 5%
Pluronic F127, (e) 7.5% F127-DA and 2.5% Pluronic F127 and (h) 10%
F127-DA. The intensity curves were multiplied by a factor for better
visualization. Fits to Eq. (11), calculated from the best-t parameters
summarized in Table 1, are shown as solid lines. (B) Normalized
scattering intensity curves of PF hydrogels with (e) 1%PEG-DA, 1.5% (h)
PEG-DA and (D) 2% PEG-DA. The intensity curves were multiplied by a
factor for better visualization. Fits to Eq. (2), calculated from the best-t
parameters summarized in Table 1, are shown as solid lines.

relative F127-DA concentration, the percentage of bound

micelles to the hydrogel increased and as a result, the distance between the micelles increased and their degree of
ordering was reduced.

Table 1
Best t parameters for the SAXS data shown in Fig. 3.
F127-DA (%w/v)

2.5%

5%

PF hydrogels with Pluronic F127/F127-DA mixtures, total blockcopolymer concentration 10 w/v%

RH ()
96.1 0.3
101.1 0.1
hb
0.289 0.002
0.253 0.002
R ()
89.4 0.4
91.9 0.2
R1cyl ()
17.0 0.3
16.4 0.3
R2cyl ()
27.2 0.5
30.6 0.4
6.25  104
6.25  104
Dq2micelle (e 6) [15]
0.106
0.106
Dq2cyl (e 6) [12]

7.5%

10%

109 1
0.205 0.003
94.6 0.8
17.2 0.3
39.1 0.4
6.25  104

125 1
0.197 0.002
85.3 0.3
18.0 0.2

0.106

0.106

6.25  104

PEG-DA (%w/v)

1%

1.5%

2%

PF hydrogels with PEG-DA

R1cyl ()
R2cyl ()
Dq2 (e 6) [12]

15.9 0.4
37.4 0.8
0.106

14.3 0.6
33.3 0.4
0.106

14.3 0.4
33.3 0.2
0.106

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MACROMOLECULAR NANOTECHNOLOGY

hypothesized structure of the nanostructured PF hydrogels

shown schematically in Fig. 4.
Cryo-TEM micrographs of crosslinked PF hydrogels containing F127-DA (Fig. 5) supported the hypothesis that the
inuence of F127-DA on the hydrogel nanostructure is pronounced only after photopolymerization of these solutions.
This is despite the fact that the nanostructure of the PF
with F127-DA was not profoundly affected in the precursor
solutions. The crosslinked specimens demonstrated distinct regions with ordered micelles, which co-existed with
regions of randomly distributed micelles. The density of
the micelles in the random regions was relatively small
(Fig. 5). No distinguishable differences in micelle size could
be observed between micrographs from different samples
with increasing concentrations of F127-DA. These

Fig. 4. Schematic representation of PF hydrogel embedded with a

mixture of Pluronic F127 and F127-DA.

observations were also in agreement with the SAXS results.

Moreover, a rough estimation of the core radius of the micelles could be obtained from the micrographs and was
found to be roughly 10 nm, which is in accordance with
the radius tted by the SAXS analysis.
One may argue that the changes to the size of the aggregated protein backbone are a consequence of the crosslinking process itself, rather than the binding of micelles to the
PF network. In order to examine this possibility, SAXS patterns of PF solutions and hydrogels crosslinked with PEGDA (rather than F127-DA) were experimentally obtained
(Fig. 3B). PEG-DA is a nonionic hydrophilic polymer chain
that does not exhibit micelle formation, but does contain
acrylate functional groups for participating in the photochemical reaction. The experimental data from these samples were tted to Eq. (2) describing scattering from a long
cylinder, where the equation was adjusted to highlight the
size distribution aspects of the data. Similarly to PF solutions with added Pluronic F127, no dissimilarities were
observed between the scattering patterns from the different solutions (data not shown). However, the scattering
curves of the hydrogels are also similar, regardless of the
degree of crosslinking (Fig. 3B). The best t parameters
were also in agreement with the trends in the scattering
curves and approximately the same for all samples (Table 1). This nding indicated that no signicant alterations
to the nanostructures occurred due to crosslinking, and
binding of micelles was indeed the reason for the observed
structural alteration in the PF/F127-DA samples.

Fig. 5. PF hydrogels with (A) 2.5% F127-DA and 7.5% Pluronic F127, (B) 5% F127-DA and 5% Pluronic F127, (C) 7.5% F127-DA and 2.5% Pluronic F127 and
(D) 10% F127-DA.

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Transient stability of micelles was investigated using

weight gain studies. The time-dependent weight gain ability of PF hydrogels crosslinked with F127-DA at different
concentrations is presented in Fig. 6A. A maximum is seen
for all the studied hydrogels at about 2 h. At longer times,
the weight decreases until equilibrium is reached. The initial rapid increase in weight could be attributed to water
uptake, which is induced by osmotic pressure and the
hydrophilic nature of Pluronic F127. The decay of weight
gain at longer times could be due to diffusion of unbound
Pluronic F127 molecules out of the hydrogel. The equilibrium weight gain depends on the ratio between F127-DA
to Pluronic F127: the larger the F127-DA percentage,
the smaller the weight gain. This observation is in line with
the suggestion that F127-DA integrates within the PF network, thus enhancing its crosslinking density and diminishing its weight gain ability. In order to further examine
the hypothesis that unbound Pluronic F127 molecules
diffuse out from the hydrogels, a mass balance was performed. The calculated amount of solids extracted during
swelling experiments, normalized to the total amount of
solids in the hydrogel, was found to increase as the relative
concentration of F127-DA in the block-copolymer mixture
decreased (Fig. 6B). This outcome could also explain the
maximum observed in Fig. 6A; after 2 h of swelling, the unbound Pluronic F127 molecules start to diffuse out from

Fig. 6. (A) Weight gain vs. time of PF hydrogels where F127-DA

concentration is () 2.5%, (N) 5%, () 7.5% and (j) 10%. (B) Extraction of
solids from hydrogels with the addition of F127-DA vs. crosslinker
concentration.

the hydrogels which results in a decrease in the swelling

ratio.
3.4. Mechanical properties
The different degrees of weight gain observed for different samples indicate that the effective degree of crosslinking depends on the concentration of F127-DA which, in
turn, is expected to alter the mechanical properties of the
hydrogels. The Youngs modulus of hydrogels which were
fully hydrated was determined using a tensile-testing
instrument. As expected, an increase in F127-DA percentage leads to higher values of Youngs modulus (Fig. 7); a
linear relationship is observed between the crosslinker
concentration and Youngs modulus values.
3.5. Effect of Pluronic F127/F127-DA ratio on fully hydrated
PF hydrogels
The concentration of F127-DA in the crosslinked PF
hydrogels affected the equilibrium weight gain of the gels
to the extent that the nal water content in the network
(after 4 days) was noticeably reduced when micelles where
immobilized within. SAXS studies were thus performed on
hydrogels with variations in Pluronic F127/F127-DA ratio
after incubation in PBS buffer at 37 C for four days, in
order to examine the differences in nanostructure that resulted when micelles were immobilized. The data was t
to Eq. (11), taking size distribution into account. The best
t parameters are summarized in Table 2. A detailed
comparison between the day 0 and day 4 hydrogels (Fig. 8,
Tables 1 and 2) revealed smearing of the inter micellar peak
for all compositions, and a shift of this peak to smaller scattering angles. This could be ascribed to the release of unbound Pluronic F127 molecules from the hydrogel, which
was also detected by mass balance analysis; released Pluronic F127 likely leads to fewer micelles with larger distances
between them. The increase in intensity of the fully hydrated
hydrogels (day 4) compared with day 0 hydrogels could be
due to an increase in the cylinders radius of gyration from
30 to approximately 50 . Consequently, data for day 4

Fig. 7. Young modulus vs. crosslinker concentration of fully hydrayed PF

hydrogels with F127-DA.

MACROMOLECULAR NANOTECHNOLOGY

3.3. Weight gain properties

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Table 2
Best t parameters for the SAXS data shown in Fig. 6.
Pluronic F127-DA

2.5%

5%

7.5%

10%

RHS ()
hb
R ()
R1cyl ()
R2cyl ()

120.2 1.5
0.180 0.002
107.4 0.2
19.4 0.2
50.6 0.2

108.9 0.3
0.230 0.002
101.7 0.2
19.5 0.1
55.4 0.2

123.9 0.1
0.199 0.001
94.6 0.4
19.0 0.2
57.2 0.2

114.4 0.5
0.241 0.002
53.7 0.3
14.8 0.2
Not available

Indeed, as the network imbibes more water, the resulting

release of Pluronic F127 molecules from the hydrogel enables the precursor molecules free interaction with one another, leading to the formation of larger proteinprotein
aggregates and subsequently larger nanostructures.

MACROMOLECULAR NANOTECHNOLOGY

3.6. Relationship between nanostructure to physical and

mechanical properties

Fig. 8. Scattering from fully hydrated PF hydrogels with a total blockcopolymer concentration of 10% (w/v), where F127-DA concentration is ()
2.5%, (N) 5%, () 7.5% and (j) 10%. Full symbols are for as-prepared
hydrogels, empty symbols for fully hydrated hydrogels. The intensity
curves were multiplied by a factor for better visualization; the same
factor was used for the curves of the as-prepared and swollen hydrogels
with the same F127-DA content. Fits to Eq. (11), calculated from the bestt parameters summarized in Tables 1 and 2, are shown as solid lines.

of hydrogels with 10% F127-DA did not give a good t to Eq.

(11), possibly due to deformation of the micelles leading to
existence of non-spherical objects.
It should also be noted that the position of the inter
micellar peak was similar for all Pluronic F127-containing
hydrogels after equilibrium weight gain, which indicates
that the distances between the micelles were similar. As
noted above, increasing the ratio of F127-DA to Pluronic
F127 reduces the number of initially formed micelles. On
the other hand, the number of unbound Pluronic F127
molecules that diffuse out of the hydrogel during the equilibrium weight gain process is smaller at higher F127-DA
percentage. Thus, the balance of these two opposing effects
may have led to an approximately constant micelle concentration in the fully swollen hydrogels.
Another structural change induced by the equilibrium
weight gain process was related to the size of the protein
backbone. The model used to analyze the data approximates the size distribution of the cylindrical protein aggregates as two distinct cylinders sizes with radii of gyration
of R1 and R2. For both day 0 and day 4 hydrogels, the value
R1 does not appear to depend on the F127-DA concentration (Tables 1 and 2), whereas R2 increased with increasing
proportions of F127-DA. For hydrogels containing any
amount of F127-DA, for example, the value of R2 increases
from about 30 at day 0 to approximately 50 following
equilibrium weight gain at day 4. This could very well be
caused by the formation of larger protein aggregates associated with an increased crosslink density of the network.

The ability of the nanostructures formed by Pluronic

F127-DA to alter the physical and mechanical properties
of the hydrogels was explored using correlation analysis
between the protein radius of gyration R2 and the Youngs
modulus at day 4 and weight gain at equilibrium. An increase in the protein radius of gyration values caused a linear increase in the Youngs modulus values and a decrease
in the weight gain values (Fig. 9A and 9B accordingly). The

Fig. 9. Protein radius of gyration vs. (A) Young modulus and (B) weight
gain at equilibrium of PF hydrogels with different F127-DA
concentrations.

O. Yom-Tov et al. / European Polymer Journal 52 (2014) 137145

4. Conclusions
A new method for the manipulation of hydrogel
mechanical and physical properties is presented through
different levels of nanostructuring. By ne tuning the
amount of F127-DA relative to Pluronic F127 molecules,
different degrees of crosslinking are achieved which inuence the hydrogels weight gain ability and Youngs modulus. SAXS and Cryo-TEM measurements revealed two
important features of the nanostructured hydrogels.
Firstly, as the hydrogel imbibes water to reach equilibrium
weight gain, the Pluronic F127 molecules leach out of the
hydrogel creating cavities and network imperfections. Secondly, proteinprotein aggregation occurs in these hydrogels during the equilibrium weight gain process, resulting
in an increased size of the protein backbone. Moreover,
SAXS and cryo-TEM data revealed closely packed micelles
at 37 C with well-ordered structures. Nevertheless, the
photochemical crosslinking caused immobilization of the
micelles in proportion to the F127-DA concentration,
which in turn caused the distance between the micelles
to increase and their degree of ordering to be reduced.
After equilibrium weight gain of the nanostructured
hydrogels, the protein backbone increased in size in proportion to F127-DA concentration. Ultimately, the nanostructures in the hydrogel impacted the physical and
mechanical properties; the equilibrium weight gain depends on the ratio between F127-DA to Pluronic F127
contents: larger F127-DA percentages leads to smaller
weight gain ability. Moreover, an increase in F127-DA percentage leads to higher values of Youngs modulus. The
equilibrium weight gain ability decreased and the Youngs
modulus linearly increased with the increase in protein
size after four days of swelling. Accordingly, it is likely that
the higher porosity and mesh size resulting from the release of unbound Pluronic F127 molecules from the fully
hydrated hydrogels had the most pronounced impact on
the hydrogels characteristics.

Acknowledgement
This research was supported by the Singapore National
Research Foundation under CREATE programme: The
Regenerative Medicine Initiative in Cardiac Restoration
Therapy (NRF-Technion).
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MACROMOLECULAR NANOTECHNOLOGY

most likely explanation for these observations ties back to

the diffusion of the block-copolymer molecules that leach
out of the hydrogel and create cavities within it; in the
fully hydrated hydrogel, the proteins radii (which are
modeled as cylinders) are able to self-aggregate thus creating longer fragments and consequently larger nanostructures. Those fragments enable high water uptake which
results in diminished Youngs modulus values. Consequently, we can conclude that alterations in nanostructure,
achieved through different degrees of crosslinking, have a
great impact on the physical and mechanical properties
of the hydrogel.

145