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Investigation of Changes in the Microscopic Structure of Anionic Poly(N‑isopropylacrylamide-co-Acrylic acid) Microgels in the Presence of Cationic Organic Dyes toward Precisely Controlled Uptake/Release of Low-Molecular-Weight Chemical Compound Takuma Kureha,† Takahisa Shibamoto,† Shusuke Matsui,† Takaaki Sato,† and Daisuke Suzuki*,†,‡ †

Graduate School of Textile Science & Technology, Shinshu University, 3-15-1 Tokida Ueda, Nagano 386-8567 Japan Division of Smart Textiles, Institute for Fiber Engineering, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan

‡

S Supporting Information *

ABSTRACT: Changes in a microscopic structure of an anionic poly(N-isopropylacrylamide-co-acrylic acid) microgel were investigated using small- and wide-angle X-ray scattering (SWAXS). The scattering profiles of the microgels were analyzed in a wide scattering vector (q) range of 0.07 ≤ q/nm−1 ≤ 20. In particular, the microscopic structure of the microgel in the presence of a cationic dye rhodamine 6G (R6G) was characterized in terms of its correlation length (ξ), which represents the length scale of the spatial correlation of the network density fluctuations, and characteristic distance (d*), which originated from the local packing of isopropyl groups of two neighboring chains. In the presence of cationic R6G, ξ exhibited a divergent-like behavior, which was not seen in the absence of R6G, and d* was decreased with decreasing the volume of the microgel upon increasing temperature. At the same time, the amount of R6G adsorbed per unit mass of the microgel increased upon heating. These results suggested that a coil-to-globule transition of the poly(N-isopropylacrylamide) chains in the present anionic microgel occurred because of efficiently screened, thus, short ranged electrostatic repulsion between the charged groups, and hydrophobic interaction between the isopropyl groups in the presence of cationic R6G. The combination of hydrophobic and electrostatic interaction between the cationic dye and the microgel affected the separation and volume transition behavior of the microgel.

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forces can also be used for molecular separation.12,13 It is wellknown that pNIPAm-based microgels can be multiresponsive when copolymerized with various functional groups. For example, poly(NIPAm-co-acrylic acid), denoted as (poly(NIPAm-co-AAc)), and poly(NIPAm-co-methacrylic acid) microgels are not only thermoresponsive but also pHresponsive owing to the presence of carboxylic acid groups, resulting in polyelectrolyte behavior.14−16 Snowden et al. investigated the adsorption of cationic lead ions using anionic poly(NIPAm-co-AAc) microgels.12 The quantity of lead ions adsorbed by the microgels increased with increasing pH because of enhanced electrostatic attractive forces. In addition, Parasuraman and Serpe reported that anionic poly(NIPAm-coAAc) microgels efficiently adsorbed the anionic azo dye Orange II.13,17,18 Their results suggested that the enhanced uptake was because of an increase in the size of the microgels upon increasing acrylic acid content. However, there have been few

INTRODUCTION Hydrogel particles (microgels) are colloidal particles with average diameters ranging from several tens of nanometers to single micrometers.1 In appropriate solvents, these particles become highly swollen with water and demonstrate stimuli responsiveness similar to bulk gels. Poly(N-isopropylacrylamide) (pNIPAm) cross-linked with N,N′-methylenebis(acrylamide) (BIS) is the most intensively studied aqueous thermoresponsive microgel.1,2 The linear pNIPAm chain is a representative thermoresponsive polymer having a lower critical solution temperature (LCST) of ca. 31 °C.3,4 Thus, pNIPAmbased microgels exhibit a volume phase transition temperature (VPTT) near the LCST of the pNIPAm chains.5 The swelling/ deswelling behavior of these microgels is suitable for various applications such as drug delivery systems,6,7 separation carriers,8,9 and sensors10 as the interactions between target molecules and the microgels can be controlled quickly. In the pNIPAm-based microgels, hydrophobic interactions between target molecules and microgels can be used to control the molecular uptake and release behavior.9,11 In addition to hydrophobic partitioning, electrostatic repulsive or attractive © XXXX American Chemical Society

Received: February 26, 2016 Revised: April 18, 2016

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DOI: 10.1021/acs.langmuir.6b00760 Langmuir XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of rhodamine 6G (R6G) (left), auramine O (center), and erythrosine (right).

even when the external environment, including that in living systems, is virtually constant (e.g., pH and temperature). The present study primarily aimed to clarify the dye uptake and release behavior of a carboxylic acid-functionalized pNIPAm-based microgel. As well, the influence of microscopic structural changes in the negatively charged pNIPAm-based microgel in the presence of a cationic dye on the separation behavior of the microgel was investigated. First, a monodisperse poly(NIPAm-co-AAc) microgel was synthesized via aqueous free-radical precipitation polymerization. The microgel was characterized using dynamic light scattering (DLS) and SWAXS techniques in the absence and presence of the target molecule. Uptake of the cationic organic dye R6G (Figure 1, left) and anionic erythrosine (Figure 1, right) by the microgel was then investigated as a function of temperature to discuss the electrostatic interaction between the target molecule and the microgel. These dyes also provide a reference because they were used to investigate the uptake behavior of pure (or nonfunctionalized) pNIPAm microgels in our previous study.20 In addition, the cationic dye auramine O (Figure 1, center) was used to confirm the effect of hydrophobicity of target molecules on the structural changes of the microgel.

reports in which the effects of both hydrophobic and electrostatic interactions among pNIPAm, polyelectrolytes, and target molecules on the molecular separation behavior of the pNIPAm-based microgels were investigated. Recently, our group investigated the relationship between the microscopic structures and functions of pNIPAm-based microgels,19−21 including the separation of organic dyes20 and swelling/deswelling or dispersing/flocculating oscillations.21 The internal microscopic structures of the pNIPAm-based microgels were investigated using small- and wide-angle X-ray scattering (SWAXS). Before that, there was no consensus about the views on the temperature dependence of correlation length (ξ), which represents the length scale of the spatial correlation of the network density fluctuations in the case of pNIPAmbased microgels. For example, in one of the frequently cited paper, Stieger et al. mentioned that the incoherent background is large and the statistical error of the experimental data is rather large in the magnitude of the scattering vector (q) range of their SANS experiments (q ≤ 1 nm−1); thus ξ of the microgel could not be extracted reliably by SANS data analysis.22 In another study, the mesh size of the network is linearly related to the hydrodynamic diameter of the microgels, showing no critical behavior.23 In our case, the SWAXS profiles were analyzed in a wide q-range (0.07 ≤ q/nm−1 ≤ 20), and this allowed us to observe the microscopic features of the microgels. As a result, ξ was precisely quantified and the characteristic distance (d*) was obtained because the two interference peaks in high-q regime were fitted by pseudo-Voigt equations. It was found that ξ exhibited a divergent-like behavior near the VPTT of the pNIPAm microgels,20 which is similar to that of pNIPAm bulk gels and chains.24,25 It was also reported that hydrophobic interactions between the hydrophobic domains corresponding to d* and both rhodamine 6G (R6G) and erythrosine, which were cationic and anionic organic dyes, respectively, influenced the uptake behavior of pure pNIPAm microgels.20 Additionally, despite the fact that small-angle scattering of Xrays (SAXS) and neutrons (SANS) are powerful techniques for structural characterization of soft materials26,27 and can provide an understanding of the internal structures of microgels (or bulk gels) in aqueous media,19−31 only few studies have focused on the characterization of functionalized pNIPAm-based microgels using SAXS or SANS techniques.32−35 Thus, a SWAXS investigation provides insights into the microscopic structures of functionalized pNIPAm-based microgels. It should be possible to determine the effects of the microscopic structures of microgels on the separation behavior of target molecules, which are useful for the development of microgels for specific applications such as separation and drug delivery. It is important to understand the relationship between the changes in microscopic structure and the separation behavior of the microgel in the presence of target molecules, which strongly interact with microgels. This is because controlled uptake and release of functionalized molecules such as drugs and proteins by the carriers are needed for delivery systems

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EXPERIMENTAL DETAILS

Materials. N-Isopropylacrylamide (NIPAm, purity 98%), N,N′methylenebis(acrylamide) (BIS, 97%), potassium peroxodisulfate (KPS, 95%), acrylic acid (AAc, 99%), rhodamine 6G (R6G, 98%), auramine O (98%), sodium chloride (NaCl, 99.5%), sodium hydroxide (NaOH, 99%), hydrochloric acid (HCl, 99%), disodium hydrogen phosphate (99%), citric acid (98%), sodium acetate (99%), and acetic acid (98%) were purchased from Wako Pure Chemical Industries and used as received. The water used for all reactions, solution preparations, and polymer purifications was distilled and then ionexchanged (EYELA, SA-2100E1). Synthesis of Poly(NIPAm-co-AAc) Microgels. The poly(NIPAm-co-AAc) microgels were prepared via aqueous free-radical precipitation polymerization using the water-soluble anionic initiator KPS. Polymerizations were performed in a 300 mL three-neck, roundbottom flask equipped with a mechanical stirrer, condenser, and nitrogen gas inlet. The initial total monomer concentration was held constant at 150 mM. A series of microgels were synthesized with varying AAc content (20 mol %, 0.432 g; 5 mol %, 0.108 g; 1 mol %, 0.022 g of the total monomer concentration) with identical BIS content (1 mol %, 0.046 g). The concentrations of NIPAm monomer were adjusted accordingly to achieve the desired total monomer content (79 mol %, 2.682 g; 94 mol %, 3.191 g; 98 mol %, 3.327 g). Monomer solutions were dissolved in 195 mL of water in the roundbottom flask and heated to 70 °C under a stream of nitrogen with constant stirring at 250 rpm. The solutions were allowed to stabilize for at least 30 min prior to initiation. Free-radical polymerization was then initiated with KPS (0.109 g) dissolved in water (5 mL). The solutions were stirred and allowed to react for 4 h. After completion of the polymerization, the microgel dispersions were cooled to room temperature. Each microgel was purified via centrifugation/redispersion with water (twice) using a relative centrifugal force (RCF) of 50000g, followed by dialysis for a week with daily water changes. Characterization of the Microgel. The hydrodynamic diameter (Dh) of the microgel was determined by DLS (Malvern Instruments B


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Figure 2. (a) Effects of pH (ionic strength = 10 mM) on the transition behavior of the pNA microgel. (b) Hydrodynamic diameter (Dh) of the pNA microgel at pH 7 and various R6G concentrations (0.1 mM (solid blue diamonds), 0.3 mM (open black squares), 0.4 mM (open orange triangles), 0.5 mM (solid red circles), 0.6 mM (open purple squares), 1 mM (open green diamonds), and 1.2 mM (open gray triangles)). (c) Dh of the pNA microgel at pH 3.5 and various R6G concentrations (0.01 mM (blue squares), 0.05 mM (green diamonds), and 0.1 mM (open red circles)). The microgel concentration was 0.1 wt % in all cases. Ltd., Zetasizer Nano S). The time-dependent scattering intensity was detected at a total scattering angle of 173°, which corresponded to a scattering vector (q) of 0.0264 nm−1 in aqueous media. Dh of the microgel was calculated from the measured diffusion coefficient using the Stokes−Einstein equation (Zetasizer software v6.12). The DLS experiments were conducted at a microgel concentration of 0.1 wt %. We confirmed that hydrodynamic diameters were able to be determined by DLS at 0.1 wt % microgel (Supporting Information (SI) Figure 1a ). Moreover, the measured correlation function data of the microgel indicated that the diffusion coefficient of the singlemicrogel was able to be proved properly because the characteristic relaxation rate, which was obtained from the field time correlation function (g1(t,q)) in the low correlation time region (SI Figure 1b,c), was proportional to q2. The samples were allowed to thermally equilibrate at the desired temperature for 10 min prior to each measurement. The autocorrelation functions were an average of 15 measurements with a 30 s acquisition time of the intensity. It meant that the scattering intensity was measured at the time corresponding to 450 s, indicating that the statistically meaningful intensity of the microgel was able to be obtained by this measurement. Moreover, Dh and the correlation functions of the microgel at pH 7 and 25 °C did not significantly change when the acquisition time extended to 60 or 120 s (SI Figure 1c). To determine the influence of pH on Dh of the microgels, a 10 mM phosphate−citric acid buffer (pH 7) and 10 mM sodium acetate−acetic acid buffer (pH 3.5) were used. The amount of copolymerized AAc was determined by potentiometric titration. The microgel dispersion in 1 mM HCl was prepared (0.1 wt %). The pH was manually lowered to 3.0, and the titration was performed with 0.1 M NaOH. Microgels in aqueous solution were observed using an optical microscope (BX51, Olympus) equipped with a digital camera (ImageX Earth type S-2.0 M ver sion 3.0.5, Kikuchi-Optical Co., Ltd.) and transferred into Vitrotube borosilicate rectangular capillaries (0.1 mm × 2.0 mm) via capillary action. SWAXS Analyses. The internal structure of the poly(NIPAm-coAAc) microgel was evaluated using SWAXS at several temperatures in the absence and presence of R6G (1 mM and 5 mM). The sample temperature was controlled using a thermostated sample holder unit (TCS 120, Anton Paar). These experiments were conducted at a microgel concentration of 1 wt %. The SWAXS intensities were determined using a SAXSess camera (Anton Paar, Graz) equipped with a block collimator and a sealed-tube anode X-ray generator (GE Inspection Technologies, Germany). The X-ray generator apparatus was operated at 40 kV and 50 mA. A Göbel mirror and block collimator provided a focused monochromatic X-ray beam of Cu Kα radiation (λ = 0.1542 nm) with a well-defined shape. All scattering intensity (I(q)) data were normalized to the same incident primary beam intensity for transmission calibration and were corrected for background scattering resulting from the capillary and solvents (10 mM phosphate−citric acid buffer, 10 mM sodium acetate−acetic acid

buffer, and R6G dissolved in 10 mM phosphate−citric acid buffer). The absolute scale calibration was accomplished using the intensity of water as a secondary standard.36 A model-independent collimation correction (desmearing) procedure was conducted based on the Lake algorithm. All fitting analyses were performed on the desmeared, absolute scattering intensities, as previously reported.19−21 Dye Uptake Experiment. The stock solutions of R6G and erythrosine (30 mM) in a 10 mM phosphate−citric acid buffer (pH 7) were prepared. A poly(NIPAm-co-AAc) microgel dispersion in a 10 mM phosphate−citric acid buffer (pH 7) was poured into a vial. The final concentration of the microgel was 0.1 wt % for all experiments. The microgel dispersion was allowed to thermally equilibrate at the desired temperature for 1 h with constant stirring at 300 rpm in an incubator (CN-25C, Mitsubishi Electric Engineering Co., Ltd.). After the solution was stabilized in the incubator, the appropriate dye stock solution was injected into the vial. The final concentration of dye was adjusted appropriately for the individual conditions (0.1 and 0.5 mM). After 5 min of exposure, the mixture was divided into three centrifuge tubes (SC-0200, Ina-Optika Co., Ltd.). A period of 5 min was adequate for the uptake study as the amount of dye uptake did not change after 5, 30, or 60 min (SI Figure 2). The mixtures were centrifuged at an RCF of 20000g to pack the microgel at the bottom of each tube. The three supernatants were carefully removed from the centrifuge tubes without disturbing the microgel pellet at the bottom, and the absorbance of each supernatant was measured using a UV−vis spectrophotometer (JASCO, V-630iRM). Dye Release Experiment. The optimal conditions for R6G uptake (40 °C, pH 7 buffer) were then used to prepare a mixture of the microgel and R6G in the same manner as described above. After the supernatant was removed from each centrifuge tube, each microgel pellet was redispersed in a different buffer solution (pH 7, pH 5, or pH 3.5) at 25 and 40 °C and placed in the centrifuge tubes at the same concentration used in the uptake experiment. Each dispersion was subsequently mixed using a thermomixer (Thermomixer R, Eppendorf) at 25 or 40 °C for 1 h. This period was selected for all microgels as complete redispersion occurred under all conditions used for the release experiments. Each mixture was then centrifuged at an RCF of 20000g, and the supernatants were removed from the centrifuge tubes. The absorbance of each supernatant was measured using a UV−vis spectrophotometer. The overall process is illustrated in SI Scheme 1.

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RESULTS Dynamic Light Scattering Analyses in the Absence or Presence of Cationic Dye. The poly(NIPAm-co-AAc) (pNA) microgel was synthesized via precipitation polymerization. Their monodispersity was confirmed by colloid crystal formation at pH 3.5 (see SI Figure 3).37,38 It can be seen C


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Figure 3. SWAXS intensities (I(q)) of the pNA microgels in a pH 3.5 (a) and 7 (b) buffer solutions. The open gray circles represent the measured intensities. The solid black curves are the best fit curves based on the sum of eqs 1 (solid red curve, except for case of pH 7), 2 (dashed blue curve), 3 (solid purple curve), and 4 (solid and dashed green curves). The gray region highlights the Ornstein−Zernike-type contribution. Upper insets show the variation of the residuals with q. The measured all profiles were shown in SI Figures 7 and 8.

In the presence of R6G at pH 7, Dh were lower than those in the absence of R6G at all temperatures. In addition, Dh of the microgel underwent a drastic change upon an increase in R6G concentration. At R6G concentrations above 1 mM, Dh suddenly increased at 38 °C (Dh ≈ 950 nm with 1.2 mM R6G) and 31 °C (Dh ≈ 800 nm with 1.5 mM R6G), indicating that the microgels were aggregated. The colloidal stability of the deswollen microgels was provided by the electrostatic repulsion as is the case with the hard particles such as polystyrene and silica particles. We infer that the colloidal stability of the deswollen microgel decreased because of electrostatic attractive forces between the cationic R6G and the anionic groups in the pNA microgel, resulting in surface charge neutralization. At pH 3.5, a similar trend was observed at R6G concentrations above 0.5 mM (Figure 2c). We also examined the influence of other ions or dyes, such as NaCl, anionic erythrosine, and cationic auramine O by DLS measurements, as shown in SI Figure 5. Dh of the microgel remained unchanged in the presence of NaCl and erythrosine, while that of the microgel decreased in the presence of auramine O at the measurement temperature and the same low concentration (0.1 mM). Note that Dh of the microgel in the presence of auramine O was greater than that in the presence of R6G at the same dye concentration (0.1 and 0.5 mM). SWAXS Analyses. To investigate the microscopic structures of the pNA microgel in the absence or presence of R6G, SWAXS was employed. An R6G solution was chosen as the solvent for the pNA microgel, and the pH was adjusted to 7 and 3.5. Note that the concentration ratio of the microgel (wt %) and R6G (mM) was 1:1 and 1:5 for the DLS and SWAXS measurements, respectively, as pNA microgel:R6G solution ratios above 1:10 caused the microgels to flocculate at the measurement temperature (20 ≤ T/°C ≤ 50 in Figure 2b). As well, the scattering intensity of R6G solution, which was above

from the image that the microgels were hexagonally ordered and in close contact with one another, indicating that they were monodispersed. Next, the temperature and pH responsiveness of the microgel were characterized using DLS. Figure 2 shows the temperature dependence of Dh of the pNA microgel in the absence and/or presence of R6G at pH 7 (Figure 2a,b), pH 3.5 (Figure 2a,c), and pH 5 (Figure 2a). At pH 7 in the absence of R6G, Dh of the microgel did not significantly change even when the temperature was increased to 50 °C (Figure 2a), suggesting that the electrostatic repulsive interactions between the carboxylic acid groups suppress the thermoresponsiveness of the microgel. Note that Dh of the microgels in a pH 5 buffer gradually decreased with increasing temperature up to 50 °C (e.g., Dh ≈ 1230 nm at 20 °C and Dh ≈ 775 nm at 50 °C). Thus, the carboxylic acid groups in the microgels were almost completely deprotonated at pH 7. However, when the charge was decreased in the pH 3.5 buffer solution, contributions from the hydrophobic interactions were expected to be dominant. A first derivative of the estimated pNA microgel volumes, d/dT· (Dh3π/6), provided a convenient measure of the critical temperature (Tc).39 Tc of the pNA microgel at pH 3.5 was determined to be 28 °C (SI Figure 3a). We also checked the hydrodynamic diameter of the microgel in the pH 7 and 3.5 solutions after adjusting pH using NaOH, HCl, and NaCl (SI Figure 4b). These hydrodynamic diameters of the microgel were close to those in the buffers at the same ionic strength (10 mM). However, in the presence of dyes, pH of solvent without the buffer was changed with varying the concentration of dyes although pH of the buffer solution was not changed. In other words, the buffer capacity was adequate for the experimental conditions of DLS, SWAXS, and the dyes uptake study. In this study, we discussed the volume transition and dye separation behavior of the microgel in the buffers. D


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Figure 4. SWAXS intensities (I(q)) of the pNA microgels in the presence of R6G (1 mM (a), and 5 mM (b)) in a pH 7 buffer solution. The open gray circles represent the measured intensities. The solid black curves are the best fit curves based on the sum of eqs 1 (solid red curve), 2 (dashed blue curve), 3 (solid purple curve), and 4 (solid and dashed green curves). The gray region highlights the Ornstein−Zernike type contribution. Upper insets show the variation of the residuals with q. The measured all profiles were shown in SI Figures 9 and 10.

length scale of the spatial correlation of the density fluctuations caused by the polymer chain. Two interference peaks were observed in the higher q-part (e.g., q ≈ 5 nm−1 and q ≈ 16 nm−1). These peaks were formally fitted using the pseudo-Voigt equation.19−21

5 mM, was larger than that of pH 7 buffer solution (see SI Figure 6). The strongly aggregated microgels precipitated in the capillary cell and was not irradiated with an X-ray, which make it difficult to obtain meaningful scattering intensity data of the pNA microgels. Figures 3 and 4 show the SWAXS scattering profiles for the pNA microgels at pH 3.5 (Figure 3a and SI Figure 7) and pH 7 in the absence (Figure 3b and SI Figure 8) and presence of R6G (Figure 4 and SI Figures 9 and 10 ). In the previously studies, the measured I(q) values of pNIPAm-based microgels were described by a sum of five contributions.19−21 In the present study, I(q) values of the pNA microgels were also nicely fitted by the same contributions excepting the case of pH 7 in the absence of R6G (Figure 3b and SI Figure 8). In the lower q-part, the forward intensity obeyed Porod’s law and originated from the interface of the microgel,40−42

I(q) ∝ q−4

IV(q) =

The scattering intensities at q < 0.3 nm the Guinier equation,19−21,24 ⎡ R 2q2 ⎤ IG(q) = IG(0) exp⎢ − G ⎥ 3 ⎦ ⎣

can be described by

(2)

where RG is the radius of gyration and IG(0) is the asymptotic Guinier intensity at q → 0. A contribution from the density fluctuations of the network in the microgel was found in the low-to-intermediate q part, which is described by the Ornstein− Zernike (OZ) equation,24,43

Ioz(q) =

Ioz(0) 1 + q 2ξ 2

1 + (q − q*)2 ξ *2

(4)

where q* denotes the peak position and ξ* is related to the size of the organized domains. The best fit was obtained using a weighted least-squares method and a residual of the fit was divided by the standard deviation at each scattering vector (Figures 3 and 4). The Porod behavior was not clearly observed when the pNA microgels were highly swollen by the presence of charged carboxylic acid groups at pH 7 in the temperature range of 25− 50 °C (Figure 3b and SI Figure 8). As a result, I(q) values of the microgels at pH 7 were able to be fitted satisfyingly without the Porod component, although the Guinier equation was necessary to fit the upturn increase of the intensity found in q < 0.3 nm−1 (SI Figure 11a,b). This term seems to arise from solidlike density fluctuation due to the inhomogeneities of cross-links.19,24 These results were due to the fact that the interface of the microgel was not well-defined. In addition, the contribution from the polymer network, which was described by the OZ equations, overlapped in the low-q regime. However, the Porod and Guinier contributions were necessary to fit the data without giving rise to systematic deviations (SI Figure 11d,e), even when the microgels were in the swollen state, for instance, below 30 °C (pH 3.5, Figure 3a), 35 °C (R6G 1 mM, pH 7, Figure 4a), and 30 °C (R6G 5 mM, pH 7, Figure 4b). Additionally, the Porod behavior became clearly visible either by lowering pH, increasing temperature, or increasing R6G concentration, for instance, above 30 °C (pH 3.5), 35 °C (R6G

(1) −1

IV(0)

(3)

where ξ is the correlation length and IOZ(0) is the asymptotic OZ intensity when q → 0. The parameter ξ represents the E


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Figure 5. (a) Temperature dependence of the correlation length (ξ) of the pNA microgel in the absence of R6G at pH 7 (solid circles) and pH 3.5 (open circles). (b) Temperature dependence of ξ of the pNA microgel in the presence of R6G at pH 7. The concentrations of R6G used in these experiments were 1 and 5 mM.

Figure 6. SWAXS intensities (I(q)) of (a) pNIPAm microgels dispersed in pure water and several poly(NIPAm-co-AAc) microgels at 25 °C in a pH 3.5 buffer solution ((b) copolymerized AAc 1 mol %, (c) AAc 5 mol %, and (d) 20 mol %, respectively). The low-q peaks of these microgels are indicated by the arrows and gray region highlights. The SWAXS profile of the pNIPAm microgels (a) adapted from ref 20.

1 mM, pH 7), and 30 °C (R6G 5 mM, pH 7) for q < 1 nm−1. These observations demonstrated that the microgel was able to be deswollen at high temperature due to protonation of the carboxylic acid groups at pH 3.5 (Figure 2a) and charge screening by R6G at pH 7 (Figure 2b), resulting in the formation of the well-defined interface. At pH 3.5, the contribution of the OZ scattering increased as the temperature increased to 28 °C and then drastically decreased at 30 °C (Figure 3a). In contrast to the case of pure pNIPAm microgels,20 the OZ contribution did not disappear for the present microgel, even above the Tc (28 °C) of pNA microgels at pH 3.5 (Figure 2a). The microgels did not completely deswell below 40 °C, indicating that the carboxylic acid groups were not completely protonated in the pH 3.5 buffer because Dh of the microgel at pH 3.5 in the presence of R6G was smaller than that in the absence of R6G (Figure 2c). In the absence of R6G at pH 7, the contribution of the OZ scattering extended over the low-to-intermediate q-range and did not significantly change in the temperature range between 25 and 50 °C (Figure 3b). However, in the presence of R6G, the OZ intensity became larger with increasing temperature up to 38 °C with 1 mM (Figure 4a) and 30 °C with 5 mM (Figure 4b), above which the OZ contribution gradually decreased with increasing temperature. Figure 5 shows ξ values for the microgel at pH 3.5 and pH 7 as a function of temperature in the absence of R6G (Figure 5a) and the presence of 1 and 5 mM R6G (Figure 5b), respectively. ξ of the pNA microgel at pH 7 increased slightly. In contrast, ξ exhibited divergent-like behavior at pH 3.5 and in the presence of R6G at pH 7, which was similar to our previous study on pNIPAm microgels.20 The maximum values of ξ for the pNA microgel

could be seen at 28 °C (Figure 5a, open circles; ξ = 4.5 nm) in a pH 3.5 buffer, 38 °C (Figure 5b, blue diamonds; ξ = 4.2 nm) in a pH 7 buffer with 1 mM R6G, and 30 °C (Figure 5b, red squares; ξ = 7 nm) in a pH 7 buffer with 5 mM R6G. The low-q peak centered at q ≈ 5 nm−1 can be attributed to interchain correlations or the domain formation of pNIPAm.19−21 The low-q peak position (q*) of pure pNIPAm microgels gradually shifted upon increasing temperature, accompanied by an increase in the peak intensity.20 This suggested that the pNIPAm main chains were in closer proximity with increasing temperature due to the hydrophobic association of isopropyl groups. It was also observed for pNIPAm-based cryogel.25 In this study, the low-q peak was not clearly observed when the SWAXS measurements were conducted on the pNA microgel dispersed in a pH 3.5 buffer below 31 °C (Figure 3a), in a pH 7 buffer (Figure 3b), and both in the presence and absence of R6G at low temperature (Figure 4). Here, to investigate the effect of AAc on the microscopic structure in terms of the scattering contribution from the interchain correlations, we also measured the SWAXS intensities of other poly(NIPAm-co-AAc) microgels containing fewer AAc than the pNA microgels (Figure 6). The low-q peak was clearly observed at 25 °C when the amount of AAc decreased or AAc was not contained in the microgels. The lowq peak was affected by the copolymerized carboxylic acid groups in the gel network despite the fact that almost all the carboxylic acid groups were protonated, indicating that a wider distribution of the distances between the two neighboring chains existed. This seems to demonstrate that the interaction between the isopropyl groups was weakened by the copolymerized carboxylic acids in the microgel. Note that the F


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Figure 7. (a) Temperature dependence of the distance (d*) of the pNA microgels at pH 7 and 3.5. R6G uptake quantity per unit gram ((b) open blue diamonds, mR6G 0.1 mM; (c) open red squares, R6G 0.5 mM) and the d* ((a) open circles, pH 3.5, solid circles, pH 7; (b) solid blue diamonds; (c) solid red squares) of the pNA microgel as a function of temperature. Each point represents an average of three replicate uptake experiments, and the error bars denote the standard deviations.

comparison (1:1, wt %/mM). To understand the influence of electrostatic interactions on R6G uptake behavior of the microgel, it was necessary to know the exact number of the charged AAc groups in the microgel. Thus, we determined the concentration of carboxylic acid groups through potentiometric measurements (SI Figure 12). R6G was added so as to achieve a 1:3 molar ratio of R6G to carboxylic acid groups when the concentration ratio of the microgel (wt %) and R6G (mM) was 1:1. R6G was also added so as to achieve a 5:3 molar ratio of R6G to carboxylic acid groups when the concentration ratio was 1:5. The amount of R6G and erythrosine taken up by the microgel as a function of temperature at pH 7 is shown in Figures 7b,c. At the same dye concentration (0.1 mM), the amount of R6G taken up by the microgel was much larger than that of erythrosine over the measured temperature range (e.g., R6G ∼6.0 mg/g and erythrosine ∼2.3 mg/g at 25 °C). Moreover, it can be seen that as the temperature increased to 40 °C, the quantity of R6G taken up by the microgel increased (e.g., ∼41 mg/g at 20 °C and ∼55 mg/g at 40 °C in 0.5 mM R6G solution, Figure 7c). In particular, with increasing R6G concentration from 0.1 mM to 0.5 mM, the temperature range in which the maximum R6G uptake was achieved shifted to a lower temperature side (0.1 mM, 25 ≤ T/°C ≤ 30; 0.5 mM, 20 ≤ T/°C ≤ 25).

pseudo-Voigt component describing the broad low-q peak was necessary because the best fit was not obtained without this component as shown in SI Figure 11c,f. It indicated that the contribution of the interchain correlations was included in all scattering profiles of the pNA microgel. The low-q peak at pH 3.5 was clearly visible at temperatures above 31 °C, in contrast to that at pH 7. As well, q* and intensity of this peak did not change significantly at 31 °C−40 °C (Figure 3a). In addition, the low-q peak at 35 and 40 °C in the presence of 5 mM R6G was much sharper than that in the absence of R6G at pH 7 (Figure 4b). The characteristic distance (d*) originates from the local packing of isopropyl groups of two neighboring chains and/or hydrogen bonds between amide groups20,25 and can be approximated by Bragg’s law (d* = 2π/q*), as shown in Figure 7. The characteristic distance, d*, of the microgels in a pH 7 buffer was much larger than that in a pH 3.5 buffer and in the presence of R6G at all temperatures. In the absence of R6G, d* of the microgels decreased with increasing temperature up to 50 °C in a pH 7 buffer (e.g., d* ≈ 7.4 nm at 25 °C and d* ≈ 3.0 nm at 50 °C) and 31 °C in pH 3.5 buffer (e.g., d* ≈ 6.3 nm at 25 °C and d* ≈ 1.1 nm at 31 °C), above which point no significant changes were seen (Figure 7a). In the presence of R6G, d* of the microgels also decreased as the temperature increased (Figures 7b,c). These figures also show the amount of dye taken up by the microgels, for which the details will be described below. The high-q peak centered at q ≈ 16 nm−1 can be ascribed to the local structure of poly(NIPAm-co-AAc) such as intrachain correlations because it correspond to ∼0.5 nm in the real space. It can be seen that the peak position shifted irregularly between q ≈ 15 and 20 nm−1 with temperature (Figures 3 and 4). The reason for this is unclear as there are several possible factors that affect the scattering intensity in the high-q regime such as hydrogen bonding between the carboxylic acid groups and/or the amide groups and the low scattering intensity of the microgel, corresponding to the measurement accuracy. Therefore, it is difficult to discuss this in more detail based on the present SWAXS data. Uptake Analyses. To clarify the effect of temperature and pH on the R6G uptake behavior of the pNA microgel, the R6G uptake was quantified. This experiment was performed under the same concentration ratio of the microgel (0.1 wt %) and R6G (0.1 or 0.5 mM) as was used for the DLS and SWAXS measurements (1:1 and 1:5, wt %/mM). We also determined the amount of anionic erythrosine taken up by the microgel for

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DISCUSSION Temperature- and pH-Induced Microscopic Structures of pNA Microgels. We used DLS and SWAXS to investigate the temperature- and pH-induced changes in the microscopic structure of pNA microgels, and it will be discussed. At pH 3.5, Dh of the pNA microgels significantly changed upon increasing temperature, which was not seen at pH 7 (Figure 2a), and ξ of the microgels exhibited divergentlike behavior at Tc (28 °C), which is similar to that observed for pNIPAm20 (Figure 5a). While Tc (or VPTT) of pure pNIPAm microgels was ∼33 °C, Tc of the pNA microgels shifted to ∼28 °C as observed in this study. Eimer et al. suggested that protons diffused from the solution into the poly(NIPAm-co-AAc) microgels, where they subsequently decrease the electrostatic repulsion either through direct association of the carboxylic acid group or counterion condensation at low pH.44 In the present study, we found that the protonated carboxylic acid groups, which can form hydrogen bonding networks with both the amide groups in NIPAm units and carboxylic acids, affected G


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between the microscopic structural changes in the microgel and the uptake behavior, the solvent conditions of the pNA microgel dispersion were adjusted to pH 7 used for DLS, SWAXS, and R6G uptake studies. Here, we discuss the interactions between the microgel and R6G, which triggered the changes in microscopic structure of the microgel and the important factors affecting the uptake behavior of the microgel. In the DLS measurements, Dh of the pNA microgel decreased in the presence of R6G, even at pH 7 (Figure 2b). The transition of the pNIPAm chains occurred at pH 7 when the concentration of R6G or auramine O increased, while that of the microgel did not significantly change in the presence of NaCl and anionic erythrosine at the same concentration (0.1 mM, SI Figure 5). We inferred that hydrophobic interactions occurred between the isopropyl groups and the tested cationic dyes. In other words, a decrease in Dh was due to the combination of hydrophobic and electrostatic interaction between the cationic dyes and the microgels, and thus the low concentration of NaCl (0.1 mM) was insufficient to deswell the pNA microgels. In addition, the hydrophobicity of R6G was higher than that of auramine O. The octanol−water partition coefficient (log Kow) of R6G used as a measurement of the hydrophobicity46,47 was higher than that of auramine O (e.g., log Kow of R6G = 2.62 and log Kow of auramine O = 1.98). As hydrophobicity of the dye increased, the hydrophobic interaction between the microgel and the dye was enhanced. Hence, the magnitude of the volume change in the presence of R6G was larger than that in the presence of auramine O at the same dye concentration upon increasing temperature from 20 to 50 °C (SI Figure 5). The value of ξ for the pNA microgel exhibited divergent-like behavior at 38 °C in the presence of 1 mM R6G and at 30 °C in the presence of 5 mM R6G at pH 7 (Figure 5b). Moreover, d* of the pNA microgels was smaller than in the absence of R6G at the individual measured temperatures (Figure 7b,c). The temperature dependence of d* for pNA microgel at pH 7 was similar to those of pNIPAm20 and pNA microgels at pH 3.5. The value of Dh was decreased with decreasing the value of d* (Figures 2b and 7b,c). Therefore, the electrostatic interaction between the carboxylic acid groups in the microgel was suppressed by the cationic R6G as a result of R6G being concentrated near the carboxylic acid groups in the microgel. Upon increasing temperature, the transition of the pNIPAm in the microgel was caused by the hydrophobic association of the isopropyl groups in the presence of R6G. However, Tc of the microgel in the presence of 5 mM R6G was higher than that in the absence of R6G at pH 3.5 (e.g., Tc ≈ 28 °C at pH 3.5 and Tc ≈ 30 °C at pH 7 and 0.5 mM R6G), although the number of R6G molecules was larger than that of the copolymerized carboxylic acid groups in the pNA microgels. Hoare and Pelton reported that poly(NIPAm-co-vinylacetic acid) microgels, which contained carboxylic acid groups predominantly on the microgel surface, formed a collapsed skin layer as cationic drugs bound and condensed the surface-localized acid residues.47 In the present study, the pNA microgel seems to have been able to form the skin layer through surface-localized R6G. The microgels did not show complete uptake of R6G at the measured temperature, although the amount of copolymerized carboxylic acid groups was larger than that of R6G (R6G was added in a 1:3 molar ratio to the carboxylic acid groups, Figure 7b). R6G Uptake and Release Behavior. On the basis of the results of DLS and SWAXS measurements, it was concluded

the transition behavior of pNIPAm chains in the pNA microgels because the NIPAm units were closer to each other in the network. In a previous study, we investigated that d* of pure pNIPAm microgels decreased as the temperature was increased to the VPTT, above which it remained constant upon a further increase of temperature. In addition, the value of d* of the pNIPAm microgels was related to the volume of the microgel.20 In the present study, d* of pNA microgels also decreased with decreasing the value of Dh (Figures 2a and 7a) when the temperature was increased from 20 to 31 °C at pH 3.5. The temperature dependence results indicate that hydrophobic association of isopropyl groups of two neighboring chains occurred. At pH 7, Dh and ξ of the pNA microgel did not change significantly when the temperature was increased, as the carboxylic acid groups were too strongly charged for the gel network to undergo a volume transition in the temperature range from 20 to 50 °C (Figures 2a and 5a). Shibayama et al. found that ξ values for charged poly(NIPAm-co-AAc) bulk gels decreased as a function of the network volume fraction.45 The absolute value of ξ exponent for the charged bulk gels was significantly smaller than that for pNIPAm bulk gels, which indicated that the network chains have a larger excluded volume due to the electrostatic repulsive interactions between the charged groups. Therefore, the electrostatic interactions were responsible for a larger excluded volume of polymer chains, as ξ of the pNA microgels did not exhibit divergent-like behavior in this study. However, Dh of the microgel slightly decreased with increasing temperature (e.g., Dh ≈ 1330 nm at 20 °C and Dh ≈ 1250 nm at 50 °C and pH 7, Figure 2a). The changes in Dh were related to the changes in d* of the microgel as it decreased, suggesting the formation of a hydrophobic domain by the NIPAm unit (Figure 7a). It indicated that the pNIPAm chains showed the coil-to-globule transition in the pNA microgel locally. As a result, ξ of the microgels was slightly increased with increasing temperature (Figure 5a). Moreover, the low-q peak corresponding to d* was not clearly visible at pH 3.5 when the amount of copolymerized AAc was increased (Figure 6). As well, at pH 7 and the individual measured temperature, the low-q peaks of pNA microgel were broader than those observed at pH 3.5 (Figure 3). It indicated that the distance between the polymer chains was expanded by the electrostatic repulsion force between AAc groups and the large distribution of the distance existed in the microgel. Note that at high temperature, i.e., above 34 °C, a scattering maximum begins to appear in the SANS data for weakly charged poly(NIPAm-co-AAc) bulk gels, which was observed in q ≈ 2 nm−1.45 This scattering maximum originates from strong concentration fluctuations due to competition between electrostatic repulsive interaction of the acrylic acid groups and hydrophobic interaction of the pNIPAm chains, leading to microphase separation. It was recently reported that microphase separation was not observed in the SANS data for poly(NIPAm-co-vinylacetic acid) microgels.32 In the present study, we could not separate a Guinier type contributions and a shoulder characterizing the microphase separation. In addition, the microphase separation of poly(NIPAm-co-1-vinylimidazole) microgels did not occur because of their small size.33 Therefore, in the near future, we will clarify the effects of size and amount of copolymerized AAc in the microgel on the volume phase transition and microphase separation using SWAXS. Microscopic Structures of the Microgel in the Presence of R6G at pH 7. To investigate the relationship H


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to describe by the theory. However, at pH 3.5, the microgels showed the volume transition behavior similar to that of pure pNIPAm microgel.20 In addition, in the presence of R6G at pH 7, the charge of carboxylic acid groups were screened by R6G, which induced the transition behavior of the pNIPAm chains in the pNA microgel. Therefore, the Flory−Rehner theory was applied to describe the transition behavior of the microgel assuming that the microgel was neutral microgel in the presence of R6G. The theoretical background and calculation of some parameters were described at the end of the SI. Indeed, the swelling curves of the pNA microgel at pH 3.5 and pH 7 in the presence of R6G (0.1 and 0.5 mM), which were determined by SI equation S1, were able to be fitted in terms of this theory (SI Figure 13). Specifically, the polymer−solvent interaction parameter, χ2, was increased from 0.32 to 0.71 when the concentration of R6G was increased from 0.1 to 0.5 mM (SI Table 1). These χ2 values were bigger than that at pH 3.5 in the absence of R6G (e.g., χ2 ≈ 0.25 at pH 3.5). This trend in χ2 can be explained by an increase of the interactions between the pNA microgel and R6G solution with increasing R6G concentration. Therefore, not only the electrostatic interaction between the carboxylic acid groups and R6G but also the hydrophobic interaction between the pNIPAm chains and R6G occurred in the pNA microgels at pH 7. Indeed, with increasing R6G concentration, Dh and d* of the microgels were decreased at the individual measured temperatures (Figures 2b and 7b,c), and Tc corresponding to the temperature dependent behavior of ξ was shifted to a lower temperature (Figure 5b). The result of the Flory−Rehner analysis of the microgels in the presence of R6G supported the proposed mechanism of structural changes in the microgel. The quantity of R6G taken up by the pNA microgels was influenced by changes in temperature. This microgel is suitable for controlling the separation of R6G through changes in temperature and pH. Figure 9 shows the quantities of R6G

that the electrostatic and hydrophobic interactions between the pNA microgel and R6G were important factors for the uptake behavior contributing to the microscopic structural changes in the microgel. Note that the uptake amount of erythrosine at pH 7 was smaller than that of R6G at the same concentration of the dyes (Figure 7b) and Dh of the pNA microgel did not significantly change in the presence of erythrosine (SI Figure 5). Therefore, the hydrophobic interaction between the microgel and erythrosine did not occur because the microgels were highly swollen by the presence of charged carboxylic acid groups in the gel networks. The amount of R6G taken up by the microgel at pH 7 increased with increasing temperature (Figure 7b,c) despite the decreasing size of the pNA microgels (Figure 2b). Moreover, d* of the microgels in the presence of R6G was decreased with increasing temperature from 20 to 40 °C when the uptake amount of R6G was increased. Considering the aforementioned data, we schematically illustrated the microscopic structural changes in pNA microgels at pH 7 in the presence of the cationic R6G at 25 and 40 °C in Figure 8.

Figure 8. Schematic diagram of the microscopic structural changes in pNA microgels at pH 7 in the presence of R6G. The represented values of hydrodynamic diameter (Dh) and characteristic distance (d*) of the microgel were obtained when the concentration ratio of the microgel (wt %) and R6G (mM) was 1:5 for DLS and SWAXS measurements.

At 25 °C in the presence of R6G, the electrostatic interaction between the carboxylic acid group and the cationic R6G became a dominant force for the uptake behavior and the changes in the microscopic structure of the microgel, resulting in a decrease of Dh and d*. On the other hand, at 40 °C (above Tc of pNA microgels), the charge of the carboxylic acid groups was screened by R6G in the first step and then the hydrophobic domains in the microgel grew, which was accompanied by the coil-to-globule transition of the pNIPAm chains. The hydrophobic R6G were adsorbed through hydrophobic interactions with the isopropyl groups in the pNA microgels, which increased the uptake amount (Figure 7b,c). Here, to check the proposed mechanisms of the structural changes in the pNA microgel in the presence of R6G, the Flory−Rehner theory was applied.30,48 In particular, the polymer−solvent interaction parameter (χ), which was called Flory−Huggins−Staverman parameter, was discussed to compare the interaction between pNA microgel and R6G, which was dissolved in the pH 7 buffer, under the different concentration of R6G. Note that the Flory−Rehner theory was applied to describe the volume phase transition of the neutral gel.30,49,50 Therefore, the transition behavior of charged microgel, such as pNA microgel at pH 7, was not appropriate

Figure 9. Amount of R6G released per unit gram of pNA microgel at various temperatures and pHs. The leftmost bar represents the R6G uptake in a pH 7 buffer solution at 40 °C, as shown in Figure 7c. Each point represents an average of three replicate experiments and the error bars denote the standard deviations.

released from the pNA microgel under various conditions. The maximum R6G uptake by the microgel occurred at 40 °C and pH 7 in the presence of 0.5 mM R6G; thus, the microgel was subjected to these conditions prior to evaluating its release behavior. As expected, the quantity of R6G released by the microgel was controlled in a stepwise manner by varying the temperature and pH. Note that the increase in the quantity of released R6G following a decrease in the pH was greater than that following a decrease in the temperature. These data demonstrated that the electrostatic interaction between the carboxylic acid group and R6G was the main factor influencing I


Langmuir the uptake and release behavior of the pNA microgel, while control of the release of R6G was achieved using the coupling effect of the hydrophobic and electrostatic interactions between the microgel and R6G.

CONCLUSIONS The combination of hydrophobic and electrostatic partitioning of the cationic R6G by the negatively charged microgel, which comprised poly(NIPAm-co-AAc), affected the separation and volume transition behavior of the microgel. The cationic R6G uptake and release behavior was clarified by relating microscopic structural information on the poly(NIPAm-co-AAc) microgel to the quantity of R6G taken up by the microgel. The internal structure of the microgel was investigated using SWAXS in the extended q-range of 0.07 ≤ q/nm−1 ≤ 20. The SWAXS intensity data for the microgel exhibited five different structural features. In particular, the microscopic structure of the microgel in the presence of R6G was characterized in terms of its ξ and d*. When the carboxylic acid groups were dissociated at pH 7, ξ of the microgel did not change significantly. On the other hand, in the presence of R6G, ξ of the microgel showed divergent-like behavior similar to that at pH 3.5. Shift of the critical temperature to a lower temperature indicated that the transition of pNIPAm segment in the microgel was induced by charge neutralization. Consequently, the quantity of R6G taken up by the microgel at pH 7 increased when the characteristic distance, d*, was decreased with increasing temperature because of the combination of electrostatic and hydrophobic interactions that occurred between R6G and the microgel. It demonstrated that the hydrophobic domains in the microgel grew. This view was quantitatively supported by the result of the Flory−Rehner analysis of the swelling curves in the presence of R6G. We believe that an understanding of the thermoresponsive behavior of charged pNIPAm-based microgels will help encourage their practical use as separation carriers for small molecules. In addition, the structural information obtained by this SWAXS investigation will be helpful for understanding the volume phase transition of charged, temperature-sensitive microgels and provide a guideline for other potential microgel applications.

ACKNOWLEDGMENTS

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b00760. Information on the confirmation of the accuracy of DLS measurement, optical microscope image of microgel, the effects of type of dyes on the hydrodynamic diameter of microgel measured via DLS, SWAXS measurement for R6G solution and the microgels, and the theoretical background of the Flory−Rehner description (PDF)

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D.S. acknowledges a Grant-in-Aid for Challenging Exploratory Research (26620177), and a Grant-in-Aid for Scientific Research on Innovative Areas (26102517) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. T. K. acknowledges a Grant-in-Aid for Japan Society for the Promotion of Science Fellows (15J1153300).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. J


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