Article

pubs.acs.org/JPCB

Single Microgels in Core−Shell Equilibrium: A Novel Method for

Limited Volume Studies
Claes Jidheden and Per Hansson*
Department of Pharmacy, Uppsala University, Box 532, SE-75123 Uppsala, Sweden
*
S Supporting Information

ABSTRACT: The interactions of cationic surfactant dodecyl-

trimethylammonium bromide and cationic protein cytochrome
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c with anionic polyacrylate microgels have been investigated in

microscopic liquid droplets by means of a micropipette
technique at ionic strength 0.01 M and pH 8. Experiments
on single microgels in solutions of limited amounts of the
surfactant provide the first evidence of microgels in a stable
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biphasic core−shell state with the surfactant partitioned to the

shell. Under the same conditions, the protein is found to
distribute uniformly in the microgels. Quantitative data in the
form of swelling and binding isotherms are presented and
compared with literature data for macrogels and with
predictions of a recent gel theory. Theory is found to be in
semiquantitative agreement with the experiments. The importance of polyion-mediated attractions between the protein
molecules is analyzed theoretically and proposed to explain the continuous but highly cooperative binding isotherms.

■ INTRODUCTION
Charged polymer networks have the capacity to absorb large
of a macroscopic gel is effectively suppressed by deformation
energy,7 whereas it can form at the surface without deforming
amounts of water to form hydrogels, soft materials with the original phase.8 However, as the gel material in the shell has
interesting mechanical properties.1 The network provides to adapt to the core, it is deformed from its preferred shape and
mechanical rigidity, making the thermodynamic state a function is therefore in a state of elevated free energy. This phase
of not only the composition, temperature, and pressure but also coexistence cost is different for collapse and swelling
the shape of the material. The equilibrium shape of a free gel is transitions, explaining the hysteresis. In particular, the
the one consistent with the lowest elastic free energy, and any coexistence cost (per chain) is largest in the first thin shells
perturbation from that shape gives rise to restoring elastic formed and decreases as the gel approaches the final relaxed
forces in the network.2 Another important property of gels is to homogeneous state.9 This explains the observation that the
respond to external stimuli by swelling or shrinking. This is transition goes to completion once the transition temperature is
taken advantage of in, for example, liver cancer therapy in reached.6,10
which responsive hydrogel microparticles (microgels) are used Volume phase transitions with hysteresis are observed also
to deliver highly cytotoxic anticancer agents.3 After admin- when charged gels are in contact with solutions of oppositely
istration to the blood vessels surrounding the tumor, the charged surfactants.11−15 In this case, the collapse is caused by
particles cause embolization by swelling, thereby starving the the accumulation of large amounts of surfactant micelles in the
tumor cells and limiting systemic spreading of the drug. gels when the concentration in the solution exceeds a critical
Large volume responses in hydrogels are often discontinuous value. The collapsed states are often very dense with ordered
transitions between swollen and collapsed states, referred to as microstructures similar to surfactant liquid crystalline
volume phase transitions.1,4,5 The driving force is essentially the phases.16−21 The swelling transition takes place at a lower
same as that for phase transitions in solutions of the polymers surfactant concentration (hysteresis)15,22 and is associated with
building up the network. However, the volume phase transition dissolution of the micelles and release of the surfactant. The
is characterized by hysteresis effects. This was first observed for surfactant concentration plays the role temperature does for
the thermally induced transition of poly(N-isopropylacryla- thermally controlled volume transitions, and similar to the
mide-co-acrylate) microgels in pure water, where the collapse latter, both the swelling and the collapse transitions proceed via
occurs at a lower temperature than the swelling transition.6 In a core−shell mechanism and go to completion once the
both directions, the new phase forms a thin shell at the surface
of the gels, which grows in thickness at the expense of the core Received: June 16, 2016
until the transition is completed. The behavior is in agreement Revised: August 12, 2016
with the notion that the nucleation of a new phase in the bulk Published: August 31, 2016

© 2016 American Chemical Society 10030 DOI: 10.1021/acs.jpcb.6b06086

J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

concentration is below and above the critical value, respectively. w = wideal + whs + wdef + wel + wtrans (2)
However, the latter is true only for gels in contact with
solutions of sufficiently large volume. In small volumes where The free energy of mixing is
the amount of surfactant available is limited, core−shell phase wideal = kT ∑ Ci ln xi , i ∈ {w , +, −, m , s}
coexistence has been found to be stable in equilibrium or at i≠p (3)
least metastable for very long times.18,23−25 So far, this has been
demonstrated only for macrogels, that is, for gels with volume with hard-sphere correction for the excluded volume
in the order of 1 mL. For such large gels, the long relaxation interaction between macroions33
time makes it difficult to assess whether the core−shell state is
an equilibrium phenomenon. Although there are strong ⎛ ϕ(4 − 3ϕ) ⎞
whs = kTCm⎜ − 1⎟
theoretical arguments in favor of equilibrium core−shell ⎝ (1 − ϕ)2
⎠ (4)
phase coexistence in spherical gels,26 this remains to be proven
experimentally for gels with short relaxation times. In the Here, kT is the thermal energy, Ci is the concentration, xi is the
present article, we address that problem by studying microgels mole fraction of species i, and ϕ is the volume fraction of
in small liquid droplets by means of a micropipette/ macroions. Segments of the cross-linked polymer chains (p)
micromanipulator technique. Single microgels are transferred have no translational entropy, and so xp is not included in the
from a surfactant-free solution to liquid droplets of surfactant sum in eq 3. Note, all molar concentrations, Ci, are expressed as
solution immersed in oil to prevent evaporation. The diameter the number of moles in a volume element in the actual state
and internal morphology of the microgels are monitored as a divided by the volume of the element in the reference state.
function of time in a fluorescence/light microscope. To help The cross-links give rise to an elastic free energy of the
visualizing the core−shell structure, small amounts of a network when a volume element is deformed from its preferred
fluorescent dye are dissolved in the micelles. The methodology shape
allows us to control the amount of surfactant available to one
kTCp
microgel and to quantify the ratio between surfactant and wdef = (2λθ2 + λr2 − 2 ln(λθ2λr) − 3)
microgel network charges in a single microgel. This makes it 2p (5)
possible to determine, for the first time, both the distribution of
micelles in microgels and the relationship between microgel where λr and λθ are the deformation ratios in the radial
volume and surfactant loading (swelling isotherm) at loading direction and the two circumferential directions, respectively,
levels intermediate between those for the fully swollen and fully and p is a material constant related to the number of segments
collapsed states, that is, at compositions where core−shell between cross-links.
phase coexistence is expected. The electrostatic free energy is given by22,26,32
The investigated microgels are in the size range of 100−300
CmlBZ2 ⎛ d(L) ⎞
μm, thus large enough to be macroscopic in a thermodynamic wel = ⎜ ⎟
sense but small enough to allow short equilibration times. To 2rm ⎝ rm + d(L) ⎠ (6a)
be able to compare with previous results for macrogels (and
microgels in large solution volumes) and with theoretical model d(L) = LC(1 − e−L / LC) (6b)
calculations, we have chosen to study the well-characterized
system of polyacrylate (PA) networks and dodecyltrimethy- Z and rm are the charge and the radius of the macroion,
lammonium bromide (C12TAB).13,17,22,26−30 Control experi- respectively, and Bjerrum length lB = e2/4πε0εrkT, where e is
ments are carried out with cytochrome c (cyt c), a positively the elementary charge and ε0εr is the permittivity of water. L is
charged protein known to distribute uniformly in PA- the thickness of the aqueous layer surrounding a macroion
microgels.31 The results are compared with theoretical positioned at the center of a spherical cell of radius rcell so that
predictions by a recent model.22,26 cmvm̅ = r3m/r3cell, where cm and vm̅ are the molar concentration and

■ THEORY
General. In a recent mean-field model,22,26,32 a hydrogel is
molar volume, respectively, of the macroion. LC is the decay
length, describing the range of attractive electric double layer
forces between the macroions. Equation 6a,6b is a general-
described as a collection of microscopic volume elements ization of a model for the interaction between planar surfaces.
containing a solution of charged polymer network, water (w), As shown elsewhere,34 the latter is identical to the strong
monovalent cations (+), monovalent anions (−), and spherical coupling theory by Moreira and Netz35 in the limit of zero
macroions (m). When the macroions are surfactant micelles, separation but extends the applicability range toward larger
the volume elements also contain free surfactant monomers (s). distances and lower electrostatic coupling strengths; for
To describe a spherical gel, a reference state is defined and the detailed discussions of the electrostatic model, see refs 34, 36.
position of each volume element is labeled by its radial distance In the case of surfactants, the micelle and free surfactant are
R from the center in the reference state. The actual state of the considered to be two different species, which can be converted
gel is represented by the function r(R), describing the radial into each other in a reversible “reaction”. The equilibrium is
distance of each volume element from the center. The free handled by assuming a constant standard free energy cost
energy of the gel is given by ΔW0mic of transforming N surfactants into a micelle. This gives a
contribution
R gel
W= ∫0 4πR2w(R ) dR
(1)
0
wtrans = ΔW micCmic (7)
Rgel is the radius of the gel in the reference state, and w(R) is ΔW0mic is dominated by the hydrophobic effect but includes all
the free energy per unit volume of the reference state. The free- nonelectrostatic contributions to the interaction free energy
energy function is a sum of independent contributions change of the process.
10031 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

Inhomogeneous Gels. The case of relevance for the 20 mL, whereafter 109 μL of TEMED was added. Right before
present work is core−shell phase separation induced by binding adding 10 mL of the mixture to a reboiler containing a
of surfactant from the solution. This was investigated in detail deareated solution of 0.09 g Span 60 (emulsifier) in 30 mL
recently by Gernandt and Hansson26 using the above model cyclohexane at 45 °C, 364 μL 0.18 M ammonium persulphate
and with parameters relevant for the system studied here. solution (initiator) was added. The reaction mixture was left for
Counterion binding to the network chains in swollen gel states 15 min, whereupon the temperature was adjusted to 60 °C.
was corrected for by solving the Poisson−Boltzmann equation After that, the reboiler was heavily stirred for 20 min with a
in cylindrical geometry, which resulted in an additional term in steady flow of nitrogen gas kept above the surface to prevent
eq 2; see eqs 1 and 9 in ref 26. For a description of the the oxygen from dissolving in the mixture. After termination of
constraints imposed on the system by the phase coexistence the reaction by addition of 40 mL methanol, the gel slurry was
and the numerical method used to determine the function r(R) removed and dispersed in additional methanol. The methanol
to be able to solve eq 1, we refer to that paper. phase was decanted off, and the gel slurry was dried in an oven
Homogeneous Gels. This case has been investigated at an airflow temperature of 60 °C (Dynamic Vapor Sorption
earlier for macroions of size and charge similar to those of cyt c Device: DVS Advantage, Particulate Systems). The dry
but without taking counterion binding to the network chains poly(acrylic acid) microgels were converted to the sodium
into account. In the present work, we correct for that by salt by first suspending in 10 mM sodium phosphate buffer (pH
treating one proportion of the counterions as bound (xb+) to the 9.0) and then rinsing with Milli-Q water to remove excess ions.
network and one as free (xf+) Finally, the product was poured a couple of times through a
pair of sieves with pore sizes of 0.300 and 0.106 mm (Test
x+ = x+b + x+f (8) Sieve Mesh Nos: 50 and 140 from Retsch) to get a smaller size
variation for easier selection and handling. The middle fraction
x+b = (1 − ξ −1)(xp − Zxs) (9) of each batch was saved and stored in sealed bottles in a
where ξ = lB/b is the charge density parameter in the refrigerator.
counterion condensation theory by Manning37,38 and b is the Concentration of Network Charges in Microgels. The
linear separation between charges on the polyion chains. procedure to determine the concentrations involved two steps.
Equation 9 states that a fraction (1 − ξ−1) of the network (1) The total sodium ion concentrations in the fractionated
charges not associated with the macroions have a counterion suspensions were determined by flame ion analysis. To degrade
bound to them. Only the free counterions contribute to the the network before analysis, a small volume of homogenized
entropy of mixing, and so eq 3 is replaced by suspension was mixed with a 2:1 mixture of nitric acid and
hydrogen peroxide and then diluted to 50 mL with pure water.
wideal /kT = Cw ln x w + C+f ln x+f + C − ln x− + Cm ln xm Analysis at two emission wavelengths (589.592 and 588.995
(10) nm) gave the following result: 209.2 and 209.8 ppm (Batch 1)
For free-swelling homogeneous gels, the total free energy and 183.9 and 187.2 ppm (Batch 2). (2) To determine the
given by eq 1 is simply W = 4πR3gelw/3. Furthermore, as only average concentration of network charges in the microgels, 20
one parameter λ is needed to define the state of the network, eq μL of suspension was mixed with 10 mM phosphate buffer (pH
5 is replaced by 8.0) in a homebuilt microscopy cell. The cell was top-filled and
sealed by a cover glass to avoid any optical phenomena due to
3kTCp curved surfaces. When all microgels had settled at the bottom
wdef = (λ 2 − 2 ln λ − 1) of the cell, microscopy pictures (Olympus BX51 with camera
2p (11)
Olympus DP73 and software cellSens dimension v.1.7.1) of the
Here, λ = Cp/cp, where cp is the molar concentration of
3
whole sample were stitched together using the Olympus
polymer segments in the swollen gel. Multiple Image Alignment software function (10% overlap).
From the free-energy function, it is straightforward to derive The total gel volume in the sample was obtained by converting
expressions for the chemical potential for all mobile species. the diameter of each microgel (measured with a circle tool) to
These are provided as Supporting Information together with a the volume of sphere. Selection criteria were set to reduce
description of how they are used to calculate the equilibrium multiple measurements of the same molecule due to overlap
state of the gels. and to include partially collapsed gels with a distinct curvature.

■ EXPERIMENTAL SECTION
Chemicals. Acrylic acid (99%), ammonium peroxide sulfate
The measurements were repeated three times with new
samples of the suspension. This resulted in mean values of
5.18 and 3.60 μL for Batches 1 and 2, respectively, which after
(98%), cyclohexane (>99%), N,N-methylenebisacrylamide combining with the sodium ion analysis resulted in an average
(99%), rhodamine B (RB) (≥95%), sodium chloride charge density of 35.3 mM (Batch 1) and 45.0 mM (Batch 2).
(>99.5%), sodium phosphate monobasic monohydrate Sample Solutions. All C12TAB and cyt c solutions were
(>98%), N,N,N′,N′-tetramethylethylenediamine (Reagent Plus prepared in 10 mM sodium phosphate buffer (pH 8.0) to
99%), all from Sigma-Aldrich, cyt c (horse heart) (>95%) from ensure that the polymer chains are fully ionized. To the
Fluka, dodecyltrimethylammonium bromide (approx. 99%) and C12TAB samples was added approximately one RB (cationic
sodium phosphate from Sigma, methanol (99.8%) from Merck, fluorescent dye) molecule per 100 surfactant molecules.
ethanol (95%) from Solveco, Span 60 from Carl Roth OHG, Determination of Critical Aggregation Concentration
and Paraffin oil from Sigma-Aldrich were all used as received. (CAC). A limited number of microgels were added to each
Microgel Synthesis. Two batches of sodium PA microgel sample in a series of phosphate-buffered C12TAB solutions of
suspensions were produced by first mixing 2.6 g of acrylic acid, different strengths. After equilibration for 1 month, the samples
11 g of 2 M NaOH solution, and 6.5 g of 80 mM N,N- were examined by microscopy. Solutions with C12TAB
methylenebisacrylamide with Milli-Q water to a total volume of concentration ≥0.78 mM contained microgels with distinct
10032 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

Figure 1. Schematic illustrations of single-microgel microscopy experiments (not drawn to scale). The cell is filled with paraffin oil to prevent
evaporation. 1. A microgel is selected and measured from a drop of gel solution. 2. The microgel is transferred into a hemispherical solution droplet
by means of a micropipette.

Figure 2. Schematic representation of the setup for bulk experiments. Leftmost is a peristaltic pump connected to a plastic pipette and a two-
compartment box creating a circular flow. A microgel reservoir is placed in compartment B and a microgel is selected with a micropipette. Both
compartments A and B are then filled with buffer, whereupon the microgel is transferred to compartment A over the middle wall. Buffer is then
removed from B until emptied, leaving compartment A filled. More buffer is added as the pump is turned on to keep compartment A filled.
Concentrated solution of cyt c is then added to the plastic pipette to create the circular flowing bulk solution, and a layer of paraffin oil is put over
compartment A to prevent evaporation.

RB-rich domains and/or fully collapsed microgels. The microgel to the hemispherical droplet from a supply of
reasoning for this was that concentrations above the CAC microgels in a second droplet positioned at the bottom of
would create dense regions of micelle-collapsed network or the cell. The microgel was selected to have a size suitable for
fully collapsed microgels whereas concentrations below CAC the volume and surfactant concentration in the droplet to give a
would create no such regions. desired molar ratio between surfactant and network charges.
Single-Microgel Microscopy. Microgels were monitored Photos were taken regularly during at least 60 min after
in an inverted microscope (Zeiss Axio Vert.A1 with cameras inserting the gel particle in the droplet. Both light and
Axiocam ICm1 and Axiocam MRc and software Zen Lite 2012) fluorescence microscopy were used to visualize the rim of the
after insertion into small hemispherical liquid droplets adhered microgels to ensure that the diameters were correctly
to the bottom of a homebuilt cell (Figure 1). The cell had walls determined. For the fluorescence, a filter module with
made of acrylic plastic attached by means of silica sealant to a excitation/emission band-pass wavelengths at 545/605 nm
16 × 16 mm2 transparent bottom window made of a glass was used. Other components of the setup were as follows:
coverslip. To prevent evaporation of the droplet, the cell was microinjector IM-11-2, pipette puller PN-31, microgrinder EG-
filled with paraffin oil. At the start of the experiment, a 400, microforge MF-900, micromanipulators M-152 and
micropipette coupled to a motor-driven microinjector (Nar- MHW-3, and glass capillaries G-1, all from Narishige.
ishige IM-300) was used to inject a small droplet of surfactant For cyt c, additional experiments were performed with the
(or protein) solution just below the oil/air surface. The microgels in contact with a solution of large volume. The
diameter was determined from microscopy images captured as experiment was conducted in a homebuilt plastic cell with two
the droplet sedimented to the bottom and converted to volume compartments, A and B, both with cover glass bottom.
by assuming a spherical shape. The latter assumption was Compartment A was connected to a peristaltic pump, P-1,
checked by observing droplets falling through oil both from the from Pharmacia Fine Chemicals to create a circular flow. Before
side and from the top. Calculations were made to ensure that entering compartment A, the tubing was connected to a plastic
surfactants adhering to the water/oil interface of the droplet pipette held at a certain height to create a self-regulating and
were of an insignificant amount compared to that of the even flow into the experiment cell. A schematic picture of the
surfactant solution. A micropipette was used to transfer a setup is shown in Figure 2. A microgel was selected from a
10033 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

supply in the nonconnected compartment B, whereupon the relaxed state after long times. In the light microscopy images
compartments were filled with enough buffer to move the gel (upper row), the micropipette can be seen to the right. Small
over the slightly lowered compartment wall. Both compart- amounts of the hydrophobic fluorescent molecule RB are
ments were then partly emptied to avoid unintentional transfer present in the samples to probe the distribution of surfactant
of microgels between compartments. The total volume was micelles. As evident from the fluorescence microscopy images
calculated by weighing in the buffer before and after enough (lower row), the probe and therefore also the surfactant
was added to the circular flow to fill compartment A and the micelles are enriched near the surface of the microgels.
tubing, bringing the bulk volume to between 9.8 and 11.4 mL. However, for some of the microgels, with β < 0.6, shells
A layer of paraffin oil was put over compartment A to avoid could not be detected by the present method, and for some, the
evaporation. Different amounts of cyt c solution were added to shells appeared not to be intact. A surfactant-rich shell is clearly
the circular flow via the plastic pipette and left for between 20 observable in the gel with β = 0.77. Careful inspection reveals
and 24 h. shells also for the gels with β = 0.54 and 0.67. For the latter

■ RESULTS AND DISCUSSION

C12TAB. To investigate equilibrium states of PA-microgels in
(but not the former), the thin shell can also be seen by light
microscopy. As a consequence of their low water content, the
shells are indeed expected to be very thin even at rather high β.
systems containing limited amounts of C12TA+ ions, single This is evident from Figure 5 showing the relative gel radius, r/
microgels were inserted into small liquid droplets by means of r0, core radius, rcore/r0, and shell thickness, (r − rcore)/r0, as
the micropipette technique in a specially constructed functions of β for spherical gels, where r0 is the gel radius for β
microscopy cell. The microgels were transferred from a = 0. The curves were calculated by assuming that the shell has
surfactant-free reference solution with the same pH and ionic the same composition as that of the fully collapsed gels at high
strength as those of the surfactant solution in the droplet (10 β and that the swelling of the core decreases linearly with β (see
mM sodium phosphate buffer, pH 8.0). The evolution of the below). According to Figure 5, the thickness of the shell in a gel
microgels was monitored and documented in a series of with r0 = 100 μm is larger than 1 μm (≈ resolution limit of the
microscopy images taken at short time intervals. Figure 3 shows microscope) for β > 0.7. This highlights the necessity of using
fluorescent probes for detection. Note, as the focal length is
comparable to the gel diameter, the fluorescence images can
easily give the impression that the entire gel contains high levels
of probe. However, for a uniform distribution of the probe, the
intensity would be highest in the center of the gel, contrary to
what is observed here.
After the initial relaxation process, the volume and
morphology of the microgels remained unchanged. The same
was observed for microgels from Batch 2. The long-time
stability of the core−shell state was tested in a separate
experiment in which a microgel from Batch 1 was first allowed
to absorb C12TAB for 5−10 s in a droplet containing 14 mM
surfactant. Because of the high concentration, the short
exposure time was enough to create a thick shell in the
microgel. The microgel was then transferred to a droplet with a
surfactant concentration equal to the CAC (0.78 mM). Even
after 24 h, the shell was intact and clearly observable (Figure 4).
As far as we are aware, the data presented here is the first
Figure 3. Volume relaxation curves for PA-microgels in microscopic evidence of microgels in a stable core−shell state induced by
droplets of aqueous solutions of C12TAB in 10 mM sodium phosphate surfactants. From a conceptual point of view, the qualitative
(pH 8.0); β-values as indicated. assessment of the biphasic gel state is the major result of the
present work.
The collected data can be used to construct a plot of V/V0
the volume of microgels from Batch 1 as a function of time for versus β. So far in the literature this type of swelling isotherm
different molar ratios between surfactant and network charges has only been available for macrogels. Here, we take advantage
in the microgels (β). The latter parameter is defined in the of the cooperative nature of the phase transition, ensuring that
following way the concentration of surfactant in the solution in equilibrium
ctot − CAC with core−shell gels is constant, which is the rationale behind
β= eq 12. The validation of this assumption is provided by binding
c PA (12)
isotherms recorded earlier for biphasic macrogels, showing that
where ctot and cPA are the total concentrations of surfactant and the free concentration is practically constant for β values
PA charges, respectively, in the droplet and CAC is the critical between 0.1 and 0.8. Figure 6 shows the result from separate
surfactant concentration for binding to the microgel (see the experiments with 30 different microgels from Batch 1, with
Experimental Section). The validity of eq 12 will be further diameters between 189 and 400 μm. Although the data points
discussed below. The volume is expressed as V/V0, that is, are rather scattered, it is clear that the volume decreases with
volume V relative to that in the surfactant-free reference increasing surfactant loading until β reaches ca. 0.8, whereafter
solution V0. The result shows that the gel volume relaxes within the volume does not change much (in absolute numbers). The
10−20 min to a new level, which decreases with increasing β. leveling out coincides, approximately, with the point where the
Figure 4 shows representative pictures of microgels in the core starts to vanish. A true homogenous state has not been
10034 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

Figure 4. Light (upper row) and fluorescence (lower) microscopy images of PA-microgels in microscopic droplets of aqueous solutions of C12TAB
in 10 mM sodium phosphate (pH 8.0). In the upper row, the micropipette holding the microgel is visible to the right of the gel in each picture. The
rightmost images: a microgel with a preformed shell after equilibration for 24 h in a droplet with a C12TAB concentration equal to the CAC (images
are not to scale with the five leftmost images). Small amounts of the hydrophobic fluorescent dye RB are present to visualize the distribution of
surfactant micelles.

Figure 6. Swelling isotherms for a PA-microgel in a solution of

Figure 5. Theoretically calculated gel radius (r), core radius (rcore), and C12TAB in 10 mM sodium phosphate buffer. The microgel volume
shell thickness (r − rcore) relative to the initial gel radius (r0) as ratio (V/V0) is plotted versus the charge ratio between the surfactant
functions of the ratio between surfactant and network charges (β) in and network (β) from experiments with microgels from Batches 1
spherical gels. (squares) and 2 (diamonds). The crosses are data for macrogels in 10
mM NaBr taken from ref 13. The thin solid line and dotted line are
just guides to the eye. The thick solid line is the prediction by the
observed in these experiments but rather wrinkled and very theory in ref 26.
dense shell formations. It might be possible that the time to
achieve a perfect spherical homogenous state is too great for
capturing the complete vanishing of the core in this β-value in Figure 6 may be slightly overestimated. For the other
experimental setup. The behavior observed here recapitulates microgels, the error introduced by eq 12 as such is expected to
that of PA-macrogels in solutions of C12TAB and 10 mM NaBr, be small. But the fact that each data point is derived from
as can be seen in Figure 6 in which data for that system are experiments with a unique microgel particle introduces an
included. The composition of the microgels in the fully uncertainty. Previous investigations of microgels prepared in
collapsed state is also similar to that of macrogels. For the the same way as the ones used here indicate that the
microgel from Batch 2 with β = 0.9, the concentration of PA composition varies little between individual microgels from
network charges is 1.7 M, which is the same as in macrogels the same batch.13,41 However, in addition to the rather large
fully collapsed by C12TAB in 10 mM NaBr solutions.13 The variation in the size of the studied particles, the value of cPA
corresponding surfactant concentration is 1.5 M, which is of the used in eq 12 is an average value and some variation around it is
order of magnitude where C12TA+ can be expected to form expected, as is the case also for other properties such as the
liquid crystalline phases.17,39,40 architecture of the polymer network. Clearly, the calculated
As already noted, for the macrogel, the free surfactant value of β would be affected by variations of CAC and cPA. A
concentration in the solution starts to increase when β exceeds comparison with the behavior of Batch 2 indicates that the
0.8. Therefore, as the free concentration is assumed to be equal network charge content has an effect. The data points for the
to CAC in eq 12, the amount of bound surfactant at the highest latter microgels (included in Figure 6), with average acrylate
10035 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

content of 45 mM, deviate markedly from the data for Batch 1

with an acrylate content of 35.3 mM. It is not clear what the
exact cause for the deviation is, but it could be related to the
synthesis being sensitive to slight variations in the experimental
conditions, resulting in an effectively larger degree of cross-
linking due to a larger fraction of cross-linkers incorporated or
larger degree of chain entanglement. This would lower the
swelling capacity of the network, which, in turn, would give rise
to a higher content in the reference state. The trend observed
here is in qualitative agreement with theoretical predictions of
the swelling of core−shell gels.18
To check the influence of the fluorescent probe, three gel
particles were investigated in the absence of RB. As can be seen
in Figure 6, in which the results for these gels are included, the
data fit the general trend. To check that the free surfactant
concentration in the solution did not exceed CAC after
surfactant had been absorbed, a microgel was removed after Figure 7. Volume relaxation curves for PA-microgels in microscopic
equilibration in a droplet and replaced by a surfactant-free droplets of aqueous solutions of cyt c in 10 mM sodium phosphate
microgel. No volume change of the latter microgel could be (pH 8.0); β-values are as indicated.
detected, indicating that the concentration remaining in the
droplet was below CAC. However, the microgel gained a faint Zctot
β=
pinkish hue, showing that at least some amount of RB was c PA (13)
taken up. This is expected because the probe molecules carry
one positive charge at the present pH and should therefore be where Z is the net charge number of the protein molecule set
slightly enriched in the gels. equal to 7. Equation 13 is considered to be accurate for all
Cyt c. Previous investigations show that cyt c is readily taken compositions investigated (see below).
up by PA-gels from solutions at low ionic strength and Figure 9 shows the terminal gel volume ratios as a function of
accompanied by strong deswelling of the gel network.42 β. Similar to the surfactant case, there is a systematic difference
However, in contrast to the case of surfactants, the degree of between Batches 1 and 2, probably related to a varying degree
binding and the volume change are continuous functions of the of cross-linking as discussed above. For both batches, the initial
concentration in the liquid. The difference can be attributed to parts of the curves show some resemblance to the ones in
the large difference in charge between the protein (+7) and the Figure 6 but the volume decreases slower with increasing β and
micelles (ca. +60).26,32 In agreement with that, cyt c has been there is a more clearly pronounced minimum in V/V0 at high β.
shown to distribute uniformly in PA-microgels at equilibrium,31 The most striking difference is however that the collapse
even at intermediate binding levels where C12TA+ forms a shell. amplitude is much smaller for cyt c than for C12TAB. This
As the binding isotherm for cyt c is continuous, states with indicates that the volume change induced per charge is smaller
intermediate degrees of binding are also stable when PA- for cyt c and explains, at least in part, why the initial slope of the
microgels are in contact with solution reservoirs of the protein isotherms is smaller.
(“bulk conditions”). These characteristics make the system The possibility to construct the swelling isotherms in Figure
suitable as a control system to check the experimental setup for 9, which are the first of their kind reported for microgels,
optical distortions and other artifacts. Moreover, combination highlights the usefulness of the drop method to systems in
of droplet and bulk condition experiments allows us to which the interaction between the microgels and the binding
determine both swelling and binding isotherms, which have substances is strong. We were not able to determine directly the
free concentration of cyt c in equilibrium with microgels in the
previously been available for macrogels only.
droplets. Nevertheless, it is possible to construct a binding
Figure 7 shows how the volume changes as a function of time
isotherm by combining the data in Figure 9 with volume data
after insertion of microgels from Batch 1 into droplets
from bulk solution experiments. For this purpose, we used a
containing different concentrations of cyt c and 10 mM sodium specially designed large solution volume cell (Figure 2). Figure
phosphate buffer (pH 8.0). Similar to the case of C12TAB, the 10 shows the volume response of 10 different single microgels
microgels relaxed to a stable state of reduced volume after determined with this setup. As can be seen, the microgel
which no further volume change took place. However, in that volume is a continuous function of the cyt c concentration, in
state, cyt c was uniformly distributed in the microgel, as can be agreement with an earlier study performed at an ionic strength
seen in Figure 8, showing microscopy images of microgels with of 40 mM.31 The microgels start to shrink already at very low
different cyt c binding ratios, where β is the charge ratio cyt c concentration. Just above 1 μM, the volume starts to
between the protein and network in the microgel. The red decrease faster but reaches a minimum V/V0 value of ca. 0.3 at
color of cyt c makes it possible to observe it with light 1.5 μM, in good agreement with the minimum in Figure 9. It
micrcoscopy, and the fact that the color is markedly stronger in can be noted that the cyt c concentration is very low in the
the center than near the surface shows that it is uniformly entire dynamic range. In fact, it can be calculated that only
distributed. Binding data for macrogels indicate that the protein between 0.7 and 1.5% is left in the solution when β is in the
is strongly partitioned to the gel at the present ionic strength. range between zero and unity. This justifies the use of eq 13 to
This allows us to calculate β from the total concentrations of calculate β in that range. To construct a binding isotherm, we
cyt c (ctot) and PA network charges (cPA) in the droplets plot the β values for each V/V0 in Figure 9 against the cyt c
10036 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

Figure 8. Light microscopy images of PA-microgels in microscopic droplets of aqueous solutions of cyt c in 10 mM sodium phosphate (pH 8.0). The
micropipette holding the microgel is visible to the left of the gel in each picture.

Figure 9. Swelling isotherms for a PA-microgel in a solution of cyt c.

The microgel volume ratio (V/V0) is plotted vs the charge ratio
Figure 10. Microgel volume ratio (V/V0) plotted vs the concentration
between the protein and network (β) for experiments with microgels
of cyt c in the solution ([cyt c]liq). Data from bulk solution
from Batches 1 and 2. The dashed and dotted lines are just guides to
experiments with microgel from Batch 1 (symbols). The solid line is
the eye. The solid line is the prediction by the homogeneous gel
the prediction of the homogeneous gel model.
theory.

concentrations in Figure 10 corresponding to the same V/V0. of binding small anions from the solution to maintain
To this end, we used a continuous function fitted to the data electroneutrality. The shape of the isotherm resembles the
points in Figure 10. The result is shown in Figure 11. Clearly, ones for PA-macrogels reported by Zezin and co-workers in the
for β in the range 0.2−1, binding takes place in a narrow range presence of 0, 40, and 80 mM NaCl.42,43 The lower part of the
of cyt c concentrations just above 1 μM, suggesting a curve is positioned at cyt c concentrations between the ones for
cooperative process, as will be further discussed below. To 0 and 40 mM, but the slope is larger. Moreover, the plateau is
the best of our knowledge, this is the first binding isotherm positioned at a higher β value than that in the curves for the
reported for binding of cyt c to PA-microgels. The cooperative macrogels. Because both types of gels are macroscopic systems
range ends quite abruptly at β ≈ 1, in agreement with the in a thermodynamic sense, the differences are expected to be
notion that further binding is associated with an entropic cost due to kinetics rather than equilibrium properties. However, the
10037 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

The point of transition to the homogeneous state is rather well

defined for both microgel batches and occurs at a lower β than
predicted by theory, especially for Batch 2. However, the
density of the collapsed state is reasonably well captured by
theory. For example, at β = 0.9, the theoretically calculated
concentration of PA-charges is 2.1 M, which can be compared
to 1.7 M for Batch 2.
In the intermediate binding range, the theoretical curve has a
distinctly concave shape, which is an effect of the elasticity of
the shell.18,26,29 For a first-order phase transition between two
states of fixed composition, the curve would be a straight line.
However, when two phases coexist in the same gel, their
composition depends on the relative amounts of them present
in the gel. In a core−shell gel, the shell network on both sides
of the border between the phases must be equally stretched in
the lateral directions, that is, λθ(core) = λθ(shell) at the
border.8,26 This means that the network in a collapsed shell
outside a swollen core is stretched in the two circumferential
Figure 11. Binding isotherm for a PA-microgel in a solution of cyt c directions and contracted in the other direction, and thus in a
constructed from the data in Figures 9 and 10 (symbols). The solid nonuniform state of elevated elastic free energy. The tendency
line represents the prediction of the homogeneous gel theory. The of the shell network to resume a uniform state gives rise to a
dotted line represents the prediction of the theory without electrostatic compressive force on the core, which responds by expelling
interactions (wel = 0). water. The swelling of the core has been found to be a nearly
linearly decreasing function of the fraction of network in the
effect can also reflect other differences between the systems, shell.18,26 For a very dense shell, the swelling isotherm then
such as the degree of cross-linking of the networks and pH of assumes the shape of a parabolic function,27 similar to the
the solution. theoretical curve in Figure 6. Whereas the latter curve does not
Comparison with Theory. C 12 TAB. Very recently describe accurately the experimental data, it captures to some
Gernandt and Hansson used the above gel model to calculate extent the shape of the curve for Batch 2. The comparison with
equilibrium properties of polyelectrolyte gels in equilibrium Batch 1 is hampered by the large scattering of the data in the
with solutions of oppositely charged surfactants.26 The model intermediate range. However, it appears that the theory
parameters were chosen to correspond to fully charged PA- overestimates the curvature of the swelling isotherm. The
networks interacting with C12TA+ in the presence of 10 mM reason for this is not clear, but one explanation is that the
monovalent salt, thus closely matching the conditions of the elasticity of the network is not properly captured by the model,
present experiments. In systems in which the volume ratio which is based on the simple Wall theory of rubber elasticity.44
between the solution and the gel is small, the model predicts Within theory, the curvature decreases with a decrease in the
core−shell phase equilibrium in spherical gels at intermediate degree of cross-linking. As this parameter is not related to the
binding ratios, in agreement with the present experiments. In actual degree of cross-linking or the structure of the real
previous reports, core−shell phase coexistence has only been network, this may be one reason for the discrepancy. Another
observed as transient states in spherical microgels undergoing explanation would be that although the theory predicts the
volume phase transition in solutions of large volume or in composition of the collapsed homogeneous state rather
macrogels of nonspherical shape. As a quantitative comparison accurately it fails to describe the interactions between the
with experiment, we have included in Figure 6 the swelling components of the deformed shells, as discussed elsewhere.26
isotherm calculated from theory (thick solid curve). The curve Cyt c. As evident from the above results for microgels as well
is new but based on data from the calculations underlying as previous investigations of macrogels, the major qualitative
Figure 10a in ref 26 obtained with the above model for difference between the behaviors of cyt c and C12TAB is that
inhomogeneous gels with the following input parameters: Cp = the former distributes uniformly in the gels and the latter
0.95 M, p = 50, LC = 1 nm, N = 60, ΔW0mic = −11.6NkT, rm = induces phase separation. Within the gel model, this is
1.7 nm, vw = vp = 1.8 × 10−5 m3/mole, v+ = v− = vs = 0, rcyl = 0.2 explained by the difference in the charge number between
nm, and w0PB = −2kT, where vi is the molar volume and rcyl and the protein and the surfactant micelles. Increasing the charge of
w0PB are the radius of the charge cylinder and a term correcting the macroions increases the strength of the polyion-mediated
for a change of reference state energy of the polyion, electrostatic attraction between them. Model calculations
respectively, in the PB-model calculations; for further details, predict that spherical macroions need to have a minimum
see ref 26. According to theory, the gel is homogeneous and charge in order for the attractions to overcome the thermal
essentially free from micelles at low β where the theoretical forces and induce core−shell phase separation.32 Interestingly,
curve has a plateau. The lack of data points in the lower binding the critical charge was found to be only slightly larger than the
range precludes us from measuring the width of the plateau, but net charge of cyt c. This suggests that the electrostatic
the positions of the isotherms in the horizontal direction attractions, although not strong enough to induce phase
suggest that shrinking does not start at extremely low β, separation, should have an effect on both the swelling and
indicating that a plateau exists. In the model, the onset of binding isotherms for cyt c. Therefore, it is interesting to
shrinking starts at β ≈ 0.07 and coincides with shell formation. compare the theoretical model predictions with the new
With increasing β, the shell grows at the expense of the core experimental data obtained here. The theoretical results for
until the collapsed homogeneous state is reached at β ≈ 0.9. C12TAB, taken from ref 26, are valid for a decay length of LC =
10038 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

1 nm, a reasonable value for the range of polyion-mediated and the largest contractive pressure comes from the electro-
attractions between the highly charged micelles.40 For the cyt c static attractions. The model overestimates V/V0 also in this
system, in which the electrostatic coupling strength is smaller, range. However, this is partly due to an underestimation of V0
we follow Gernandt and Hansson by equating the decay length because the theoretically calculated concentration of PA at β =
with the Debye length, lD. Instead of eq 6b, we use the 0 is larger (58 mM) than that in the microgels from both
following relationships batches. In fact, at high β, the calculated degree of swelling is
lD intermediate between the values for Batches 1 and 2. For
d (L ) = (1 − e−L / lD) example, at the minimum of the curves, the PA concentration is
2 (14a)
0.14 M (theory), 0.12 M (Batch 1), and 0.18 M (Batch 2). For
0.304 nm comparison, the value for macrogels in the absence of salt is
lD = (25 °C, 1:1 electrolyte)
csalt /M (14b) 0.16 M.42 The reswelling at high β has two origins. At the
minimum, it is caused by the excluded volume interaction
Equation 14a is different from that in ref 32, but for small L, among the proteins,29 and when β exceeds 1, the swelling
d(L) ≈ L/2 in both cases. The present form is preferred increases also because protein counterions become incorpo-
because it is consistent with the expression used for C12TAB. rated.
As an improvement of the theory in ref 32, the present Turning to the binding isotherms in Figure 11, it is clear that
calculations correct for counterion binding to the network the theoretical curve starts to level out at much lower β-values
chains (cf. eqs 8−10). The theoretical results are included in than in the experiments. This happens when the excluded
Figures 9−11 (solid lines). All parameters used in the volume interaction between the macroions becomes important.
calculations are given in Table 1. The first thing to notice is
The repulsion increases the chemical potential of the macroion,
but the effect from that on the binding isotherm is small.
Table 1. Parameters in the Homogeneous Gel Model
Instead, an analysis of the free-energy changes involved reveals
PA, cyt c network, macroiona that the leveling out is related to counterion entropy. In the
Z +7 +10, +25, +50 cooperative binding range, the mole fraction of counterions in
rm 1.5 nm 2 nm the gel is nearly constant despite the fact that counterions are
vw 0.03 nm3 0.03 nm3 released into the solution. This is because the gel volume
p 50 37 decreases rapidly with increasing β in this range. However,
b 0.25 nm 0.6 nm when the excluded volume interactions dominate the repulsive
Cp 1.4 M 7.336 M osmotic pressure, the gel volume decreases slower and
csalt (1:1) 0.01 M 0 therefore the mole fraction of counterions decreases. This
a
System Monte Carlo (MC)-simulated by Edgecombe and Linse.46 decreases the gain in entropy from releasing counterions into
the solution, which has a dramatic effect on the partitioning of
that β and V/V0 are continuous functions of the free macroions between the gel and the solution. This is in
concentration in the solution, in agreement with the experi- agreement with the previous results showing that binding
ments (see Figures 10 and 11). However, the rapid change in a isotherms are hypersensitive to the gel volume response
narrow concentration range is an effect of the electrostatic characteristics.45 As the volume response results from a delicate
attractions, as evident from comparison with the binding balance of forces, it is not clear whether the failure of the model
isotherm obtained by setting wel = 0 (purely entropic model) in the upper binding range is due to an overestimation of the
included in Figure 11 (dotted line). The theory captures most repulsive contributions to the osmotic pressure or from an
features observed experimentally, including the minimum in the underestimation of the attractive contributions.
swelling isotherm (Figure 9) in the region close to the protein/ Macroions in Model Network. Figure 12 compares swelling
network charge stoichiometry as well as the concentration in isotherms calculated from the homogeneous gel model with the
the solution where cooperative binding starts. The quantitative MC simulations by Edgecombe and Linse.46 The simulated
agreement is far from perfect, of course, but it should be systems consist of negatively charged networks, positively
pointed out that the curves are calculated using physically charged spherical macroions, and small monovalent cations (β
realistic parameters (Table 1) without parameter fitting. < 1) or anions (β > 1) (but no salt). The components are
Therefore, it is particularly rewarding that the initial part of
immersed in a medium with dielectric constant equal to that of
the binding isotherm is in good agreement with the
water at 25 °C. The polymer chains consist of 37 charged beads
experiments. The model underestimates the deswelling in the
of radius 0.2 nm connected by harmonic springs (freely
lowest binding range, where (in the model) the volume for a
given β is determined mainly by a balance between the swelling jointed). All chains have the same length and are connected
forces due to the entropy of the small ions and the contractive into a perfect network with a diamond-like topology. Shown are
elastic forces in the network. Therefore, a likely explanation is the results for systems containing 60, 24, and 12 macroions of
that the network model fails to capture the responsiveness of radius 2 nm and charge numbers +10, +25, and +50,
the most swollen microgels. However, in this range, all respectively. The systems are too small for investigations of
contributions to the osmotic pressure vary slowly with the gel macroscopic phase separation, but the well-defined network
volume and so the position of the balance point is sensitive also structure, free from entanglements and loose ends, and the
to contributions which are small in absolute numbers. other characteristics make them suitable for testing the
In the contracted states at high degrees of binding both the homogeneous gel model. The theoretical curves in Figure 12
repulsive and attractive forces are much stronger than in the (solid lines) were calculated with the parameters given in Table
swollen state. Here, the entropy of mixing the proteins and the 1. The decay length, LC, used in eq 6b was calculated from the
network chains contribute substantially to the swelling pressure, charge and radius of the macroion according to
10039 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

layer of counterions (here the beads in the chains), as discussed

in detail elsewhere.34,36 Parameter b, describing the distance
between charges along the polymer chains, is not constant in
the simulated system, in which the bond length is the result of
the interaction between neighboring beads. However, rather
independently of conditions, the average bond length was
found46 to deviate little from the value given in Table 1. The
elastic model requires the specification of Cp, the concentration
of segments in a state of zero elastic free energy, which is not
available from the simulations. The value in Table 1 was
calculated within the model with knowledge of the equilibrium
swelling in a reference state where the beads are uncharged and
no macroions are present.47 In Figure 12, the swelling ratio, Q,
is defined as the volume relative to the volume in the reference
state and β is the ratio between macroion and network charges.
Despite the correction for counterion binding, the model
overestimates the swelling of the macroion free network (β =
0) by a factor of 2.0. The reason is not clear but probably
reflects a shortcoming of the network elasticity model,
including the uncertainty of the Cp-value. As our main concern
is the effect of the interaction between the macroions and the
network, we have normalized the calculated volumes with that
factor. As can be seen, the model captures the volume change
induced by the macroions quite well, which is rewarding,
considering the large variation of the macroion charge and that
the model is free from fitting parameters. However, with
increasing macroion charge, the swelling to the left of the
minimum gel volume goes from being underestimated to being
overestimated and the swelling to the right of the minimum is
consistently underestimated. For the macroion with the highest
charge, there is a jump transition between two homogeneous
states of equal free energy, not seen in the MC simulations
(unless it occurs between two data points). But snapshots from
the simulations reported in ref 46 show an increased tendency
for clustering of the macroions with increasing charge, which
may be a manifestation of the same effect. Recall also that for
highly charged macroions the homogeneous state becomes
metastable against the core−shell phase separated already at
low β, but it is neglected here for the sake of comparison with
the MC simulations.

■ CONCLUSIONS
The results of the present article demonstrate the usefulness of
the droplet technique for investigations of microgels. The single
most important result is the evidence of core−shell phase
separation in spherical PA-microgels in equilibrium with
C12TAB solutions. Earlier the phenomenon has been observed
only in macrogels, where the question of equilibrium or not
always remains due to the long relaxation times, or as
thermodynamically unstable states in microgels undergoing
volume phase transition in solutions of large volume. The
Figure 12. Swelling isotherms for a model system of charged network capacity of the technique to provide new quantitative
+ spherical macroions in water. Symbols are from MC simulations information is demonstrated by the determined swelling
taken from ref 46. The solid lines are calculated from the isotherms, showing for the first time how the volume of single
homogeneous gel model with the parameters in Table 1. Macroion PA-microgels depends on the ratio between surfactant and
charge numbers (Z) are as indicated. polyelectrolyte charges in the gel. The swelling isotherms
resemble those reported earlier for nonspherical macrogels. It is
2rm also shown, with PA/cyt c as a demonstrator system, how the
LC =
Z (15) combination of droplet experiments and bulk solution
experiments can be used to determine both swelling and
Equation 15 expresses the idea that the range of the ion binding isotherms for systems not displaying a volume phase
correlation attraction between the macroions should be transition. Comparison between experiments and theoretical
comparable to the distance between the ions in a compressed model calculations shows that the recently developed gel theory
10040 DOI: 10.1021/acs.jpcb.6b06086
J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

is in semiquantitative agreement with the results for both cyt c (14) Nilsson, P.; Hansson, P. Regular and Irregular Deswelling of
and C12TAB, where the compositions of the collapsed Polyacrylate and Hyaluronate Gels Induced by Oppositely Charged
homogeneous states at high binding ratios and the high Surfactants. J. Colloid Int. Sci. 2008, 325, 316−323.
cooperativity of cyt c binding are particularly well captured. The (15) Sasaki, S.; Koga, S.; Imabayashi, R.; Maeda, H. Salt Effects on
the Volume Transition of Ionic Gel Induced by the Hydrophobic
calculations suggest that the latter feature is an effect of
Counterion Binding. J. Phys. Chem. B 2001, 105, 5852−5855.
polyion-mediated attractions between the protein molecules. (16) Dembo, A. T.; Yakunin, A. N.; Zaitsev, V. S.; Mironov, A. V.;
Furthermore, comparison with MC simulations of spherical Starodubtsev, S. G.; Khokhlov, A. R.; Chu, B. Regular Microstructures
macroions in a model network shows that the gel model is in Gel-Surfactant Complexes: Influence of Water Content and
capable of covering a wide range of electrostatic coupling Comparison with the Surfactant Structure in Water. J. Polym. Sci.,
strengths.

■
Part B: Polym. Phys. 1996, 34, 2893−2898.
(17) Hansson, P. Surfactant Self-Assembly in Polyelectrolyte Gels:
ASSOCIATED CONTENT Aggregation Numbers and Their Relation to the Gel Collapse and the
*
S Supporting Information Appearance of Ordered Structures in the NaPA/C12TAB System.
The Supporting Information is available free of charge on the Langmuir 1998, 14, 4059−4064.
(18) Hansson, P.; Schneider, S.; Lindman, B. Phase Separation in
ACS Publications website at DOI: 10.1021/acs.jpcb.6b06086.
Polyelectrolyte Gels Interacting with Surfactants of Opposite Charge.
Additional theoretical relationships for the calculation of J. Phys. Chem. B 2002, 106, 9777−9793.
the equilibrium state of homogeneous gels: (A) (19) Khandurina, Y. V.; Dembo, A. T.; Rogacheva, V. B.; Zezin, A. B.;
contributions to the chemical potential of all species in Kabanov, V. A. Structure of Polycomplexes Composed of Cross-
the gel; (B) chemical potentials in the liquid; (C) Linked Sodium Polyacrylate and Cationic Micelle-Forming Surfac-
equilibrium conditions (PDF) tants. Polym. Sci. 1994, 36, 189−194.

■
(20) Khokhlov, A.; Makhaeva, E. E.; Philippova, O. E.; Starodubtzev,
S. G. Supramolecular Structures and Conformational Transitions in
AUTHOR INFORMATION Polyelectrolyte Gels. Macromol. Symp. 1994, 87, 69−91.
Corresponding Author (21) Zhou, S.; Burger, C.; Yeh, F.; Chu, B. Charge Density Effect of
*E-mail: [email protected]. Tel: +46184714027. Polyelectrolyte Chains on the Nanostructures of Polyelectrolyte-
Notes Surfactant Complexes. Macromolecules 1998, 31, 8157−8163.
The authors declare no competing financial interest. (22) Gernandt, J.; Hansson, P. Hysteresis in the Surfactant-Induced

■
Volume Transition of Hydrogels. J. Phys. Chem. B 2015, 119, 1717−
ACKNOWLEDGMENTS 1725.
(23) Filippova, O. E.; Makhaeva, E. E.; Starodubtsev, S. G.
This work was financially supported by the Swedish Research Interaction of the Low Cross-Linked Gel of Diallyldimethylammo-
Council (grant no. 621-2011-4325).

■
nium Bromide with Sodium Dodecylsulfate. Polym. Sci. 1992, 34, 602−
606.
REFERENCES (24) Khandurina, Y. V.; Rogacheva, V. B.; Zezin, A. B.; Kabanov, V.
(1) Tanaka, T. Phase Transitions of Gels. In Polyelectrolyte Gels; A. Interaction of Cross-Linked Polyelectrolytes with Oppositely
Harland, R. S., Prud’homme, R. K., Eds.; American Chemical Society: Charged Surfactants. Polym. Sci. 1994, 36, 184−188.
Washington, DC, 1992; Vol. 480, pp 1−21. (25) Starodubtsev, S. G. Influence of Topological Structure of
(2) Hill, T. L. An Introduction to Statistical Thermodynamics, 2nd ed.; Polyelectrolyte Networks on Their Interaction with Oppositely
Addison-Wesley Publishing Co.: Reading, MA, 1962. Charged Micelle-Forming Surfactants. Vysokomol. Soedin., Ser. B
(3) Lewis, A. L.; Holden, R. R. DC Bead Embolic Drug-Eluting Bead: 1990, 32, 925−930.
Clinical Applications in the Locoregional Treatment of Tumors. (26) Gernandt, J.; Hansson, P. Surfactant-Induced Core/Shell Phase
Expert Opin. Drug Delivery 2011, 8, 153−169. Equilibrium in Hydrogels. J. Chem. Phys. 2016, 144, No. 064902.
(4) Hill, T. L. Some Statistical Mechanical Models of Elastic (27) Göransson, A.; Hansson, P. Shrinking Kinetics of Polyacrylate
Polyelectrolytes and Proteins. J. Chem. Phys. 1952, 20, 1259−1273. Gels in Surfactant Solution. J. Phys. Chem. B 2003, 107, 9203−9213.
(5) Tanaka, T. Collapse of Gels and the Critical Endpoint. Phys. Rev. (28) Hansson, P. Self-Assembly of Ionic Surfactant in Cross-Linked
Lett. 1978, 40, 820−823. Polyelectrolyte Gel of Opposite Charge. A Physical Model for Highly
(6) Matuso, E. S.; Tanaka, T. Kinetics of Discontinous Volume-Phase Charged Systems. Langmuir 1998, 14, 2269−2277.
Transition of Gels. J. Chem. Phys. 1988, 89, 1695−1703. (29) Hansson, P. Surfactant Self-Assembly in Oppositely Charged
(7) Onuki, A. Paradox in Phase Transitions with Volume Change. Polymer Networks. Theory. J. Phys. Chem. B 2009, 113, 12903−12915.
Phys. Rev. A 1988, 38, 2192−2195. (30) Nilsson, P.; Unga, J.; Hansson, P. Effect of Salt and Surfactant
(8) Sekimoto, K. Temperature Hysteresis and Morphology of Concentration on the Structure of Polyacrylate Gel/Surfactant
Volume Phase Transitions of Gels. Phys. Rev. Lett. 1993, 70, 4154− Complexes. J. Phys. Chem. B 2007, 111, 10959−10964.
4157. (31) Johansson, C.; Hansson, P. Distribution of Cytochrome C in
(9) Doi, M. Gel Dynamics. J. Phys. Soc. Jpn. 2009, 78, No. 052001. Polyacrylate Microgels. Soft Matter 2010, 6, 3970−3978.
(10) Tomari, T.; Doi, M. Hysteresis and Incubation in the Dynamics (32) Gernandt, J.; Hansson, P. Core−Shell Separation of a Hydrogel
of Volume Transition of Spherical Gels. Macromolecules 1995, 28, in a Large Solution of Proteins. Soft Matter 2012, 8, 10905−10913.
8334−8343. (33) Carnahan, N. F.; Starling, K. E. Equation of State for
(11) Hansson, P. Interaction between Polyelectrolyte Gels and Nonattracting Rigid Spheres. J. Chem. Phys. 1969, 51, 635−636.
Surfactants of Opposite Charge. Curr. Opin. Colloid Interface Sci. 2006, (34) Hansson, P.; Bysell, H.; Månsson, R.; Malmsten, M. Peptide-
11, 351−362. Microgel Interaction in the Strong Coupling Regime. J. Phys. Chem. B
(12) Kokufuta, E.; Suzuki, H.; Yoshida, R.; Kaneko, F.; Yamada, K.; 2012, 116, 10964−10975.
Hirata, M. Volume Collapse of a Cationic Poly(Ethyleneimine) Gel (35) Moreira, A. G.; Netz, R. R. Binding of Similarly Charged Plates
Induced by the Binding of Anionic Surfactants. Colloids Surf., A 1999, with Counterions Only. Phys. Rev. Lett. 2001, 87, No. 078301.
147, 179−187. (36) Bračič, M.; Hansson, P.; Pérez, L.; Zemljič, L. F.; Kogej, K.
(13) Nilsson, P.; Hansson, P. Ion-Exchange Controls the Kinetics of Interaction of Sodium Hyaluronate with a Biocompatible Cationic
Deswelling of Polyelectrolyte Microgels in Solutions of Oppositely Surfactant from Lysine: A Binding Study. Langmuir 2015, 31, 12043−
Charged Surfactant. J. Phys. Chem. B 2005, 109, 23843−23856. 12053.

10041 DOI: 10.1021/acs.jpcb.6b06086

J. Phys. Chem. B 2016, 120, 10030−10042
The Journal of Physical Chemistry B Article

(37) Anderson, C. F.; Record, T. M. Polyelectrolyte Theories and

Their Applications to DNA. Ann. Rev. Phys. Chem. 1982, 33, 191−222.
(38) Manning, G. S. Limiting Laws and Counterion Condensation in
Polyelectrolyte Solutions I. Colligative Properties. J. Chem. Phys. 1969,
51, 924−933.
(39) Balmbra, R. R.; Clunie, J. S.; Goodman, J. F. Cubic
Mesomorphic Phases. Nature 1969, 222, 1159−1160.
(40) Svensson, A.; Piculell, L.; Karlsson, L.; Cabane, B.; Jönsson, B.
Phase Behavior of an Ionic Surfactant with Mixed Monovalent/
Polymeric Counterions. J. Phys. Chem. B 2003, 107, 8119−8130.
(41) Nilsson, P.; Hansson, P. Deswelling Kinetics of Polyacrylate
Gels in Solutions of Cetyltrimethylammunium Bromide. J. Phys. Chem.
B 2007, 111, 9770−9778.
(42) Zezin, A. B.; Rogacheva, V.; Skobeleva, V.; Kabanov, V.
Controlled Uptake and Release of Proteins by Polyelectrolyte Gels.
Polym. Adv. Technol. 2002, 13, 919−925.
(43) Skobeleva, V. V.; Rogacheva, V. B.; Zezin, A. B.; Kabanov, V. A.
Collapse of Swollen Gel Network and Phase Transition in a Weakly
Cross-Linked Polyelectrolyte Gel Upon Its Interaction with
Oppositely Charged Proteins. Dokl. Phys. Chem. 1996, 347, 52−55.
(44) Wall, F. T.; Flory, P. J. Statistical Thermodynamics of Rubber
Elasticity. J. Chem. Phys. 1951, 19, 1435−1439.
(45) Johansson, C.; Gernandt, J.; Bradley, M.; Vincent, B.; Hansson,
P. Interaction between Lysozyme and Colloidal Poly(NIPAM-co-
acrylic acid) Microgels. J. Colloid. Int. Sci. 2010, 347, 241−251.
(46) Edgecombe, S.; Linse, P. Monte Carlo Simulations of Cross-
Linked Polyelectrolyte Gels with Oppositely Charged Macroions.
Langmuir 2006, 22, 3836−3843.
(47) Edgecombe, S.; Linse, P. Volume fraction of beads in the
reference state: 0.052. Lund University, Sweden. Personal communi-
cation, 2009.

10042 DOI: 10.1021/acs.jpcb.6b06086

J. Phys. Chem. B 2016, 120, 10030−10042