Dependence of the swelling behavior of a pH-responsive PEG-modified nanogel on the cross-link density

Abstract

We report pH-responsive structural changes in PEG-modified (PEGylated) nanogels, as determined by using small-angle X-ray scattering and dynamic light scattering. The size of the nanogels discontinuously increased at a lower pH than the pKa of the nanogels. This size increase was attributed to the swelling of the core part of the nanogel upon a change in pH. The swelling behavior was dependent on the cross-link density of the core. When the cross-link density was low, the core swelled greatly with preserving the polydispersity in size and maintained a constant shape; however, when the cross-link density was high, the core swelled minimally, and only the polydispersity increased. This difference in swelling behavior is discussed in terms of the inhomogeneous structural distribution of cross-links in the core.

Full text

ORIGINAL ARTICLE
Dependence of the swelling behavior of a
pH-responsive PEG-modified nanogel on the
cross-link density
Goshu Tamura
1,2
, Yuya Shinohara
1,2
, Atsushi Tamura
3,6
, Yusuke Sanada
2,4
, Motoi Oishi
3
, Isamu Akiba
2,4
,
Yukio Nagasaki
3,5
, Kazuo Sakurai
2,4
and Yoshiyuki Amemiya
1,2
We report pH-responsive structural changes in PEG-modified (PEGylated) nanogels, as determined by using small-angle X-ray
scattering and dynamic light scattering. The size of the nanogels discontinuously increased at a lower pH than the pK
a
of the
nanogels. This size increase was attributed to the swelling of the core part of the nanogel upon a change in pH. The swelling
behavior was dependent on the cross-link density of the core. When the cross-link density was low, the core swelled greatly with
preserving the polydispersity in size and maintained a constant shape; however, when the cross-link density was high, the core
swelled minimally, and only the polydispersity increased. This difference in swelling behavior is discussed in terms of the
inhomogeneous structural distribution of cross-links in the core.
Polymer Journal (2012) 44, 240–244; doi:10.1038/pj.2011.123; published online 23 November 2011
Keywords: drug delivery system; PEGylated nanogel; pH-responsive swelling; small-angle X-ray scattering
INTRODUCTION
Nanoparticle-based drug delivery systems, including liposomes,
polymeric micelles and other nanoparticles, have received growing
attention with respect to their clinical application to cancer
chemotherapy. These formulations improve therapeutic efficacy
while mitigating the severity of the side effects of tumoricidal pay-
loads, through altering the drug’s pharmacokinetics and pharmaco-
dynamics.
1–4
Because the bio-distribution of intravenously
administered nanoparticles is largely dependent on their size and
surface properties, numerous surface modification techniques have
been developed to regulate distribution within the body for therapeu-
tic treatment.
5–7
In this regard, poly(ethylene glycol) (PEG) modifica-
tion by the covalent coupling of hydrophilic PEG to pharmaceutical
materials, often referred to as ‘PEGylation,’ represents a crucial strategy
for prolonging the blood circulation time of delivery vehicles; densely
grafted PEG-tethered chains minimize nonspecific interactions with
serum proteins and the endothelia that line the blood vessels, through
entropic repulsion.
8
This technology was developed from pioneering
work on the chemical attachment of PEG to proteins,
9
and its efficacy
has been demonstrated for polymeric micelles constructed from PEG
hydrophobic block copolymers,
10
for which the hydrophobic chains
aggregate to form a spherical core domain in aqueous solutions.
Sterically stabilized PEG-modified (PEGylated) nanoparticles, such as
stealth liposomes and core-shell polymeric micelles, can escape recogni-
tion by immune system responses, such as those of the reticuloendothe-
lial system, resulting in long blood circulation times and preferential
accumulation in tumor tissues through the enhanced permeability and
retenti on effect.
11,12
To further enhance the therapeutic efficacy in
targeted tissues, as well as to reduce the side effects in normal tissues,
the selective release of therapeutic payloads in response to external
stimuli, such as pH, temperature, light irradiation, and reaction with
specific molecules or enzymes, is employed via drug nanocarriers.
Among various external stimuli, pH is of greatest interest because a
drastic decrease in pH is known to occur in various tissues and
organelles such as endosomes/lysosomes, extra-tumoral envir onments,
and inflamed regions, where the pH is lower than that of normal tissue
andthebloodstream(whichhaveapHB7.4);
13–16
thus, the develop-
ment of pH-responsiv e drug nanocarriers should be an effective strategy
to facilitate the clinical use of nanocarrier-based drug delivery systems.
Nagasaki et al. have developed core-shell-structured and pH-
responsive PEGylated nanogel particles consisting of a cross-linked
poly[2-(N,N-diethylaminoethyl) methacrylate] gel and tethered PEG
chains.
17–22
These nanogel particles may be applicable not only for
drug delivery systems but also for various therapeutics and pathog-
Received 26 May 2011; revised 31 August 2011; accepted 9 October 2011; published online 23 November 2011
1
Department of Advanced Materials Science, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan;
2
Japan Science and Technology Agency (JST)
CREST, Chiyoda, Tokyo, Japan;
3
Department of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan;
4
Department
of Chemistry and Biochemistry, University of Kitakyushu, Kitakyusyu, Fukuoka, Japan and
5
Satellite Laboratory of International Center for Materials Nanoarchitechtonics, National
Institute for Materials Science, University of Tsukuba, Tsukuba, Ibaraki, Japan
6
Present address: Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University.
Correspondence: Dr Y Shinohara, Department of Advanced Materials Science, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba
277-8561, Japan.
E-mail: yuya@k.u-tokyo.ac.jp
Polymer Journal (2012) 44, 240–244
&
2012 The Society of Polymer Science, Japan (SPSJ) All rights reserved 0032-3896/12
www.nature.com/pj

nomony, such as small interfering RNA delivery, cancer photothermal
therapy, magnetic resonance imaging and apoptosis probes, that are
used to monitor responses to cancer therapy. For all of these applica-
tions, the poly[2-(N,N-diethylaminoethyl) methacrylate] gel core of
the nanogels acts as a reservoir for anticancer drugs, small interfering
RNAs, and metal nanoparticles through hydrophobic interactions,
electrostatic interactions and coordination bonds with poly[2-(N,N-
diethylaminoethyl) methacrylate] segments. The therapeutic effects of
these nanogel derivatives have been well demonstrated,
22
but the
detailed structural changes that occur upon the release of drugs, for
example, have not yet been clarified.
In the present study, we sought to investigate the detailed pH-
dependent structural changes in a PEGylated nanogel. In previous
studies, pH-dependent changes in the hydrodynamic radius of nano-
gel particles were visualized with dynamic light scattering (DLS);
17–22
the size of nanogel particles increases under acidic conditions and
decreases under alkaline conditions. Although DLS is widely used to
characterize particle size and size distribution, estimates using DLS are
often affected by many factors, including particle shape and surface
structure, as discussed in Results and discussion. To characterize the
detailed structure of the nanogel studied herein, particularly its core,
we utilized small-angle X-ray scattering (SAXS) and DLS. SAXS is a
powerful technique used to determine the structure of soft materials
and has been successfully applied to determine the structure of
core-shell shaped polymeric micelles.
23–28
In this paper, we first
describe the pH-dependent swelling behavior of the PEGylated
nanogel and then demonstrate the dependence of pH-responsive
swelling on the cross-link density of PEGylated nanogels.
MATERIALS AND METHODS
Materials
PEGylated nanogels with different cross-link densities were synthesized by the
emulsion copolymerization of 2-(N, N-(diethylamino)ethyl methacrylate
(DEAMA; Wako, Japan), ethyleneglycol dimethacrylate (EGDMA; Wako,
Japan), and heterobifunctional a-acetal-o-vinylbenzyl-PEG macromonomer
(acetal-PEG-VB)
22
in the presence of potassium persulfate (KPS; Wako, Japan)
as an initiator. The details of the synthesis procedure are described elsewhere.
17–22
The number-averaged molecular weight, M
n
,andM
n
/M
w
of the acetal-PEG-VB
were 7870 and 1.07, respectively, where M
w
is the weight-averaged molecular
weight. The PEGylated nanogels consisted of a DEAMA core and a shell
composed of acetal-PEG-VB. The core chain was cross-linked by EGDMA
monomers; thus, the amount of EGDAMA defined the cross-link density of the
core. The molar ratio of DEAMA and acetal-PEG-VB was held constant at
[DEAMA]: [acetal-PEG-VB]¼98.8: 1.2 for all samples. The cross-link density
of the nanogels was defined as the molar percentage of EGDMA to the sum of
DEAMA and acetal-PEG-VB. The sample code is shown in Table 1. All samples
were purified by ultrafiltration (cutoff molecular weight: 200 000, Advantec,
Tokyo, Japan) using methanol to remove the unreacted starting reagents,
followed by ultrafiltration using deionized and distilled water to replace the
solvent. The obtained nanogel solution was used as a stock solution, the
concentration of which was determined by weighing the sample before and
after lyophilization.
The chemical composition of PEGylated nanogels was determined by CHN
elemental analysis using a series II CHNS/O Analyzer 2400 (Perkin Elmer,
Waltham, MA, USA). The results are shown in Table 2. The measured CHN
compositions were consistent with the feed molar compositions, indicating that
the PEGylated nanogels were synthesized as designed.
Titration measurement
The degree of protonation, a,andpK
a
of the PEGylated nanogels were
measured with potentiometric titration. The stock solutions of PEGylated
nanogel (7.8 mg) were mixed with NaCl (23.4 mg) and 0.01 N HCL (6.0 ml);
then, their volumes were adjusted to 40 ml using distilled water to produce a
final NaCl concentration of 10 m
M. This solution was titrated with 0.01 N
NaOH containing 10 m
M NaCl at using an automatic titrator (DL-25, Mettler-
Toledo, Zurich, Switzerland) at 298 K. The titrant was added in quantities of
0.05 ml at intervals of 30 s. From the obtained titration curves and the molar
amounts of sample and titration solution, pH-a curves were calculated.
DLS measurement
The hydrodynamic radius (R
h
) of the nanogel and its standard deviation (dR
h
)
were measured by DLS (Zetasizer Nano ZS, Malvern Instruments, Worcester-
shire, UK) equipped with a 4-mW He-Ne-ion laser (wavelength¼633 nm). The
DLS measurements were performed at 298 K at a detection angle of 1731,and
the values of R
h
and dR
h
/R
h
were estimated with the cumulant method.
29
The
sizes of the nanogels at a concentration of 100 mg/ml under various pH
conditions were measured. The pH values of the gels were adjusted by adding
10 m
M of TRIS (tris(hydroxymethyl)aminomethane) solution and 10 mM of
TRIS-HCl (Trizma: tris(hydroxymethyl)aminomethane hydrochloride) solution.
SAXS measurement
SAXS measurements were performed at BL03XU
30
and BL45XU,
31
SPring-8
(Hyogo, Japan). An R-AXIS VII imaging plate detector (Rigaku, Tokyo, Japan)
was used to record the scattered X-ray intensity I(q) as a function of q.The
scattering vector q is expressed in terms of the X-ray wavelength l and the
scattering angle 2y as q¼4p/l sin y. Two ion chambers located upstream and
downstream of the sample were used to measure the X-ray transmittance of the
sample. To reduce parasitic scattering, a specially designed vacuum cham-
ber
27,32
was used. The solution samples were sonicated for 1 min using a
homogenizer (UH-50, SMT, Tokyo, Japan) a few minutes before SAXS
measurements; this procedure enabled us to effectively remove large aggregates
that would have caused undesired noise in the scattering intensity profiles at
low angles. Then, the samples were placed into a quartz capillary cell
(j¼2.0 mm, Hilgenberg GmbH, Malsfeld, Germany) and sealed with an epoxy
resin. The wavelengths, the sample-to-detector distances, and the exposure
times were 0.090 and 0.15 nm, 4.0 and 3.5 m, and 30 and 300 s at the BL03XU
and BL45XU, respectively. The value of q was calibrated by the diffraction peak
of silver behenate.
32
To cover a wide q-range, the SAXS intensity profiles at the
BL03XU (0.02oqo0.1 nm
1
) and BL45XU (0.05oqo1nm
1
) were com-
bined. All measurements were performed at room temperature.
RESULTS AND DISCUSSION
Protonation of nanogel upon pH change
Figure 1 shows the pH-a curves of the PEGylated nanogels. The degree
of protonation of CD1, CD2 and CD5 at pH¼8.0 was approximately
zero. By contrast, the degree of protonation of CD1 and CD2 at
Table 1 Sample code and the molar ratio of DEAMA, acetal-PEG-VB
and EGDMA
DEAMA Acetal-PEG-VB EGDMA
CD1 98.8 1.2 1
CD2 98.8 1.2 2
CD5 98.8 1.2 5
Table 2 Results of CHN analysis of PEGylated nanogels
Feed Observed
C[%] H[%] N[%] C[%] H[%] N[%]
CD1 61.06 9.84 4.96 61.28 9.81 5.16
CD2 61.05 9.82 4.93 60.97 9.33 5.20
CD5 61.04 9.76 4.82 60.01 9.20 4.80
Swelling behavior of a pH-responsive nanogel
G Tamura et al
241
Polymer Journal

pH¼5.8 was B90% and that of CD5 was B80%. From the pH-a
curves, the pK
a
values of CD1, CD2 and CD5 were evaluated to be 6.9,
6.9 and 6.6, respectively; the results show that the pK
a
values of the
nanogels slightly shifted toward an acidic pH with the increase in the
cross-link density of the core.
Increase in nanogel size upon pH change
The pH dependence of R
h
and dR
h
/R
h
on the cross-link density of the
PEGylated nanogel is shown in Figure 2. The values of R
h
drastically
changed around the pK
a
of each sample; the sizes of PEGylated
nanogel particles increased at a pH level lower than the pK
a
. This
tendency is consistent with a previous DLS measurement of similar
nanogel particles.
22
The values of R
h
at a higher pH were B40 nm
regardless of their cross-link density, whereas those at a lower pH
decreased with the increase in cross-link density. This result indicates
that the cross-link density of the core affects the increase in nanogel size.
Results of SAXS measurement
Figure 3 shows the SAXS intensity profiles of the PEGylated nanogels
and their corresponding fitting curves at pH 5.8. and pH 8.0. Several
shoulders are recognized in the intensity profiles, although their shape
is not clear. This result indicates that the size and shape of the
PEGylated nanogels were moderately monodispersed and that the
PEGylated nanogels show a sphere-like structure. At pH 8.0, the
scattering intensity profiles are almost identical to each other, whereas,
at pH 5.8, they are significantly different. A comparison of the
intensity profiles shows that CD1 and CD2 increase in size upon a
change in pH from 8.0 to 5.8 compared with CD5; these results
support the above-mentioned DLS results.
To obtain more detailed structural information, we fitted the SAXS
intensity profiles by employing several kinds of structural models and
found that a concentric core-shell sphere model provides the best-fit
curve. The function describing concentric core-shell spheres is as
follows:
IðqÞ¼Nr
2
e
Dr
c
V
c
Fq; R
c
ðÞ+Dr
s
V
total
Fq; R
total
ðÞ½
2
;
where N is the number of spheres and r
e
is the classical electron radius.
Dr
c
and Dr
s
represent the contrast in electron density between the
core and the shell and that between the shell and solvent, respectively.
The sizes of the core and shell are defined by the radius of the core R
c
and that of the entire particle R
total
. V
c
and V
total
are the volume of the
core and the entire particle, respectively. F(q) is the form factor of a
sphere, which is described by the following equation:
Fðq; rÞ¼
3sinðqrÞqr cosðqrÞ½
qrðÞ
3
:
The polydispersity of nanogel particles is taken into account by
assuming that the core volume has a Gaussian distribution, whose
variance is evaluated with the standard deviation of the particle radius,
dR
c
. In this study, the scattering contrast of the shell, Dr
s
, is much
smaller than that of the core, Dr
c
; thus, the assumption that only the
core size is polydispersed is guaranteed when interpreting the present
scattering intensity profiles. The fitting curves agree well with the
experimental data, indicating that the present core-shell model is fairly
reasonable.
Swelling behav ior of core upon pH change
The fitted values of R
c
and dR
c
/R
c
are shown in Figure 4. The radius of
the core, R
c
, increases upon a change in pH from 8.0 to 5.8, for all the
samples, although the increase in the radius of CD5 is much smaller
than that of the other gels; this result indicates that the core of a
PEGylated nanogel is swollen upon a change in pH. The origin of this
swelling behavior is reasonably attributed to the protonation of the
core as follows. According to the titration measurement (Figure 1), the
Figure 1 Dependence of degree of protonation on pH: CD1 (circles), CD2
(triangles), and CD5 (squares). A full color version of this figure is available
at Polymer Journal online.
Figure 2 Dependence of R
h
(open symbols) and dR
h
/R
h
(closed symbols) of
PEGylated nanogels on pH: CD1 (circles), CD2 (triangles), and CD5
(squares). A full color version of this figure is available at Polymer Journal
online.
Figure 3 SAXS intensity profiles of CD1, CD2, and CD5 at pH 5.8 (left) and
pH 8.0 (right): CD1 (circles), CD2 (triangles), and CD5 (squares). The lines
show fitting curves. A full color version of this figure is available at Polymer
Journal online.
Swelling behavior of a pH-responsive nanogel
G Tamura et al
242
Polymer Journal

nitrogen atoms of DEAMA in the core were not protonated under
alkaline conditions (pH 8.0). By contrast, most of the nitrogen atoms
in the core were protonated under acidic conditions (pH 5.8) by the
influx of H
+
ions; the ions may have attached themselves to an amine
group of DEAMA. Thus, the electrostatic repulsion in the cores at pH
5.8 increases compared with that at pH 8.0 for all of the cross-link
densities. This increase in electrostatic repulsion may be a dominant
driving force of the swelling of the core upon a change in pH.
Figure 4 shows that the degree of the size-increase highly depends
on the cross-link density; the core size at pH 5.8 increases as the
cross-link density decreases. This tendency is somewhat peculiar
when one recalls the fact that the number of nitrogen atoms that
functions as a source of repulsive forces is similar over the entire
sample. The results of titration measurements (Figure 1) indicate
that most of the nitrogen atoms were protonated at pH 5.8; thus,
the repulsive force due to the protonation should be on the same
order. It should be noted that counter ions such as Cl

neutralize
the effect of protonation to a certain extent and that this might
depend on the cross-link density; nevertheless, the above discussion
holds qualitatively. The discrepancy between the core sizes of the
samples with the different cross-link densities is reasonably explained
by considering the competition between repulsive forces (electrostatic
repulsion forces) and attractive forces, such as that manifested by the
rubber elasticity of core polymers. The force originating from rubber
elasticity generally depends on the cross-link density. Let us assume
that (i) the number of core polymers, not counting the cross-linker
(EGDMA), is constant independent of the cross-link density, (ii) the
core of the swollen state is incompressible, and (iii) the interaction
between partial chains (the chain between neighboring cross-link)
can be ignored; then, the free energy density of elasticity is simply
proportional to the number of partial chains. This qualitatively shows
that the attractive force, or the force opposing the swelling, increases
with the cross-link density. The preceding discussion is purely
qualitative and requires further rigorous experimental and theoretical
study; thus, we omit the quantitative discussion in the present paper.
It should be noted that the polydispersity of the core size obtained
by SAXS measurement (Figure 4, lower panel) is independent of the
cross-link density at pH 8.0, while it increases with the increase of
cross-link density at pH 5.8. The polydispersity is, in principle,
independent of the pH conditions for each specimen, as is the case
for dR
h
/R
h
(Figure 2). The change in polydispersity can be explained
in terms of the structural inhomogeneity of the core. In CD1, the
number of cross-links in the core is relatively small; this causes the
region that obstructs homogeneous swelling due to the presence of
cross-links to be relatively small. In this case, the swelling is expected
to develop homogeneously and the polydispersity of the core size does
not change after swelling. In the case of CD5, the number of cross-
links in the core is relatively large; the core certainly has regions where
swelling develops and regions that do not swell. In this way, the core
inhomogeneously swells both in shape and size; as a result, the
polydispersity of CD5 greatly increases upon swelling. CD2 has a
moderate number of cross-links, and its polydispersity at pH 5.8 is
thus an intermediate value between the polydispersities of CD1 and
CD5. The dependence of R
c
on the cross-link density supports this
notion. In CD5, the core size barely changes upon a change in pH
from 8.0 to 5.8, while the polydispersity greatly increases; this indicates
that tiny parts of the core swell or deform upon a change in pH,
whereas the core does not swell as a whole. This results in an
inhomogeneous shape and size distribution of the core.
From the above discussion, it can be concluded that the distribution
of cross-links is not homogeneous, particularly in CD5. This situation
is schematically shown in Figure 5. The inhomogeneous structural
distribution of cross-links affects the difference in swelling behavior
between CD1, CD2 and CD5 in addition to the competition between
the repulsive and attractive forces in the core described previously.
This structural model provides a good explanation of the difference
between the pH dependence shown by SAXS and DLS. Hydrodynamic
radii are estimated from the diffusion behavior of particles in DLS,
whereas the actual topological structure can be measured by SAXS.
The hydrodynamic radii will be highly affected by the presence of a
Figure 4 Dependence of R
c
(upper) and dR
c
/R
c
(lower) on pH: CD1 (circles),
CD2 (triangles), and CD5 (squares). A full color version of this figure is
available at Polymer Journal online.
Figure 5 Schematic view of swelling behavior of a PEGylated nanogel. When
the cross-link density is low (CD1), the number of cross-links is small; thus,
the swelling develops rather homogeneously. When the cross-link density is
high (CD5), the number of cross-links is large, which results in an
inhomogeneous deformation of the nanogel. A full color version of this figure
is available at Polymer Journal online.
Swelling behavior of a pH-responsive nanogel
G Tamura et al
243
Polymer Journal

shell, and the effect of core polydispersity (dR
c
/R
c
)onthepolydis-
persity of the hydrodynamic radius (dR
h
/R
h
) will be smeared; thus, the
polydispersity of the hydrodynamic radius hardly depends on a change
in pH. In the case of CD5, the increase in core polydispersity is likely
to increase the associated drag forces in solution, which leads to an
increase in the estimated hydrodynamic radius; thus, the hydrody-
namic radius increases with a change in pH from 8.0 to 5.8, whereas
the core size barely changes.
CONCLUSIONS
In this study, we investigated the structure of PEGylated nanogels with
SAXS and DLS. Nanogel cores were observed to swell upon a change
from alkaline to acidic pH conditions. A higher cross-link density in
the core prevents the nanogel from homogeneously swelling, that is,
the core size hardly increases and the polydispersity of the nanogel
particle size changes. At a lower cross-link density, the core consider-
ably increases in size upon a change in pH with homogeneous
swelling. This swelling behavior may have a key role in the
pH-induced controlled release of drugs. Further studies such as the
in situ observation of drug release from pH-responsive nanogels using
time-resolved SAXS will help us to clarify the mechanism of drug
release, which will lead to the development of high-performance drug
delivery system systems.
ACKNOWLEDGEMENTS
The SAXS experiments were performed under the approval of the SPring-8
Proposal Advisory Committee (2009B1202 and 2009B7200). We appreciate the
support of Dr H Masunaga and Dr K Ito (JASRI) when performing the
experiments.
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