Partitioning of Small Molecules in Hydrogen-Bonding Complex

May 9, 2017 - Complex coacervation of polymers can be a route to the compartmentalization of aqueous solutions. Presented here is a study of the hydro...
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Partitioning of Small Molecules in Hydrogen-Bonding Complex Coacervates of Poly(acrylic acid) and Poly(ethylene glycol) or Pluronic Block Copolymer Mengmeng Zhao,† Seyed Ali Eghtesadi,‡ Mahesh B. Dawadi,§ Chao Wang,† Shuyue Huang,† Amy E. Seymore,∥ Bryan D. Vogt,† David A. Modarelli,§ Tianbo Liu,‡ and Nicole S. Zacharia*,† †

Department of Polymer Engineering, ‡Department of Polymer Science, and §Department of Chemistry, University of Akron, Akron, Ohio 44325, United States ∥ Department of Chemistry, Lorain County Community College, Elyria, Ohio 44035, United States S Supporting Information *

ABSTRACT: Complex coacervation of polymers can be a route to the compartmentalization of aqueous solutions. Presented here is a study of the hydrogen-bonded complex coacervation of poly(acrylic acid) and poly(ethylene glycol) or Pluronic block copolymers and the ability of these coacervates to encapsulate various ionic and nonionic dyes as well as a pharmaceutical compound within them. The formation of complex coacervate driven by hydrogen bonding is studied as a function of both pH and salt content with turbidimetry and isothermal calorimetry. Small-angle X-ray scattering shows the presence of micelles within Pluronic containing coacervate materials formed with a Pluronic block copolymer concentration higher than its critical micelle concentration. Although dyes generally partition to the coacervate phase, in the absence of salt, dyes that are able to hydrogen bond with the coacervate components are better incorporated into the coacervate. It is observed that the addition of salt to the polymer solutions increases the hydrophobicity of the environment within the coacervate, increasing the ability to sequester dye molecules for which there is no hydrogen bonding with the coacervate components. These materials are characterized with UV−vis spectroscopy, dynamic light scattering, zeta potential measurements, isothermal calorimetry, small-angle X-ray scattering, and fluorescence spectroscopy.

1. INTRODUCTION Complex coacervation, a liquid−liquid phase separation caused in some cases by mixing solutions of colloids or macromolecules,1 can be used as a route to compartmentalize chemical reactants, such as small organic molecules, within microscale water-filled environments. This compartmentalization or partitioning can be of interest for multiple reasons; loading polymer particles with pharmaceutical compounds for drug delivery purposes,2 removing dilute contaminants from aqueous solutions,3−5 and also for origin of life studies.6 Regarding this last topic, partitioning of chemicals within aqueous solutions is a promising route toward synthetic cellularity as well as relevant to origin of life studies. Progress on synthetic cellular systems has been made through the use of membrane-bounded microcompartments in the form of selfassembled bilayer vesicles,7−9 polymer capsules,10,11 and inorganic vesicles.12,13 However, such processes are often limited by the high impermeability of the membrane, which may inhibit the possibility of continuous activity within the vesicle due to the limitation of mass transfer. In this regard, the spontaneous phase separation of polymer liquid microdroplets in aqueous solution, here termed complex coacervation,1 provides a simple alternative procedure for compartmentalization without a membrane. The formation of complex © XXXX American Chemical Society

coacervates of macromolecules has been suggested to have been the original way in which solution compartmentalization took place before the evolution of phospholipid bilayer membranes.14,15 The surface tension between the polymerrich and water-rich phases is generally low, potentially facilitating the transfer of small molecules into the coacervate phase.16 Specific intermolecular interactions such as electrostatics and hydrogen bonding between macromolecules in aqueous solution have been long known to give rise to the formation of soluble complexes or to phase separation, either liquid− liquid (coacervation) or liquid−solid (precipitation) depending on stoichiometry and other factors.17−19 The liquid−liquid phase separation results in the formation of the “complex coacervate” or a dense, fluid, polymer-rich phase and a very dilute polymer-deficient phase, existing in equilibrium with one another. Complex coacervates formed using two oppositely charged macromolecules20−22 or macromolecules and surfactants23−25 via electrostatic interactions are possible as well as those between pairs of hydrogen-bonding polymers.19 HydroReceived: December 31, 2016 Revised: April 26, 2017

A

DOI: 10.1021/acs.macromol.6b02815 Macromolecules XXXX, XXX, XXX−XXX

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were centrifuged for 3h at 8000 rpm (Allegra X-30R Centrifuge, Beckman Coulter). After centrifugation, the supernatant was removed using a micropipet, and the coacervate phase was left in the bottom of centrifuge tubes. UV−vis measurement (Agilent 8453 spectrophotometer) was used to determine either the dye content of the supernatant or the dye content of the coacervate via dissolution of the coacervate phase. The sequestration was calculated as eq 1.

phobic interactions will also contribute to the coacervate formation, especially for hydrogen-bonded systems. Our previous study4 shows that complex coacervates formed with oppositely charged polyelectrolytes have the ability to sequester cationic dye, specifically methylene blue, through electrostatics or π−π interactions. In that work it was shown that complex coacervate with a combined ability to form electrostatic and π−π interactions shows a much higher sequestration ability for aromatic dye molecules than those capable of electrostatic intercations only. These preliminary studies may give insight into better designing polymeric coacervate-based materials for drug delivery or personal care products. Here, we present a study of the partitioning of four dyes methylene blue (MB), Janus green B (JGB), bromothymol blue (BtB), and 8-anilino-1-naphthalenesulfonic acid (ANS)into two different hydrogen-bonded coacervate systems, formed from poly(acrylic acid) (PAA) in combination with either Pluronic F-127 (F127) or poly(ethylene glycol) (PEG). The process of dye sequestration into the PAA-F127 and PAA-PEG coacervate phases was studied using UV−vis and fluorescence spectroscopy. The influence of added NaCl and KCl on the coacervate formation as well as the sequestration of MB and BtB was also studied. The addition of salts to hydrogenbonding complexes can have different effects. For example, added salt can increase the number of hydrogen bonds formed between poly(acrylic acid) and poly(acrylamide).26 In this work it was observed that in the absence of salt hydrogen-bonding interactions are required between the dye and the polymers to ensure high levels of sequestration, but the addition of salt enhances the hydrophobicity of the coacervate environment, also improving sequestration of dyes that do not form specific interactions with the coacervate phase.

sequestration (%) =

amount of dye in coacervate phase total amount of feeding dye in system

× 100%

(1)

2.4. Release of Dyes out from Coacervates. The PAA-F127 and PAA-PEG coacervates with different dyes were prepared as mentioned above. After 24 h stirring, samples were centrifuged for 3 h at 8000 rpm. After centrifugation, the supernatant was removed using a micropipet, and the coacervate phase was left in the bottom of centrifuge tubes. The dye content of the supernatant (0 × removal) was measured using UV−vis measurement. Then the supernatant was replaced by Milli-Q water with the same pH. After 24 h stirring, the coacervate system was centrifuged again and dye concentration of the supernatant was determined (1 × removal). This procedure was then repeated (2 × removal). 2.5. Dynamic Light Scattering Measurement. PAA (40 × 10−3 M) and F127 (40 × 10−3 M) or PAA (40 × 10−3 M) and PEG (40 × 10−3 M) solutions were mixed at equal volume to give a final solution of either PAA-F127 with a concentration of 20 × 10−3 M PAA and 20 × 10−3 M F127 or PAA-PEG with a concentration of 20 × 10−3 M PAA and 20 × 10−3 M PEG. After mixing, 1 M HCl was added to these samples to adjust pH and form coacervate. For each sample, DLS measurements were done over 24 h using a Zeta PALS instrument. Dynamic light scattering was also used to determine the formation of micelle of block copolymers including F127, P84, and P123 at certain concentrations. 2.6. FTIR. Fourier transform infrared (FTIR) spectra were acquired by a Bruker Alpha-P FIIR spectrometer with transmission mode. The samples were tested immediately after 3 h centrifugation, and all measurements were done at room temperature. 2.7. Fluorescence Spectroscopy. Steady state emission spectra of PAA-F127/ANS and PAA-PEG/ANS microdroplet dispersions were measured using a Horiba FluoroMax 4 spectrofluorometer with an excitation wavelength of 350 nm. Fluorescence emission was measured from 400 to 650 nm in quartz cuvettes for microdroplet dispersions prepared with 20 × 10−3 M PAA and 20 × 10−3 M F127 or 20 × 10−3 M PAA and 20 × 10−3 M PEG with the addition of 0.1 × 10−3 M ANS and different concentration of NaCl at pH 3.25. All fluorescence was attributed to ANS within the microdroplets as ANS fluorescence was quenched in water. 2.8. Small-Angle X-ray Scattering (SAXS). The structure of the coacervates was elucidated using SAXS (Rigaku S-MAX 3000) that employed Rigaku MicroMax-002+ microfocus sealed tube X-ray source to generate Cu Kα radiation (λ = 1.54 Å) with a divergence of 4.8 mrad and a focal spot of 20 × 20 μm. The sample-to-detector distance was 1.5 m, which corresponds to a probed momentum transfer vector, q, of 0.10−1.7 nm−1 (real space distances of 3.7−63 nm). The water-rich solutions of the individual polymer (PAA or F127) solution and PAA-F127 coacervates were contained in boronrich glass capillaries (Charles Supper Co., wall thickness ∼0.01 mm, inner diameter ∼1.0 mm) that were sealed with wax for the SAXS characterization. The scattering data were collected for 15 min. The scattering profiles from the samples containing F127 were fit to the form factor, P(q), for monodisperse hard spheres. In this case, the correlation between the F127 micelles was negligible, and thus the contribution of the structure factor, S(q), to the total scattered intensity, I(q), was insignificant for the samples examined.27 Here, I(q) for the solution of monodisperse spherical particles with smooth surfaces and uniform scattering length density is shown in eq 2.28

2. MATERIALS AND METHODS 2.1. Materials. Poly(acrylic acid) (PAA, Mw = 50 000 g/mol) was purchased from Polysciences, Inc. Poly(ethylene glycol) (PEG, Mw = 6800 g/mol) was purchased from Scientific Polymer Products, Inc. Pluronic F-127 (F127, PEO100-PPO65-PEO100) was purchased from Sigma-Aldrich. Pluronic P-123 (P123, PEO19-PPO69-PEO19) and P84 (F127, PEO19-PPO43-PEO19) were obtained from BASF. Dyes including methylene blue (MB), Janus green B (JGB), bromothymol blue (BtB), and 8-anilino-1-naphthalenesulfonic acid (ammonium salt, ANS) were purchased from Sigma-Aldrich. All water was dispensed from a Milli-Q water system at a resistivity of 18.2 MΩ·cm. All these materials were used as received without further purification. 2.2. Turbidity Measurement. Turbidity was used to qualitatively measure the extent of coacervate formation as a function of pH. Turbidity measurements were performed using a 2 cm path length fiber-optic probe colorimeter (Brinkmann PC 950) at 420 nm. Turbidity was reported as 100 − T%, where T corresponds to the transmittance. Stock solutions of PAA (40 × 10−3 M), F127 (40 × 10−3 M), and PEG (40 × 10−3 M) were prepared separately and then mixed at equal volumes to give 50 mL total volume with initial pH of 3.8 for both PAA-F127 and PAA-PEG. Addition of 1 M HCl into the prepared PAA-F127 or PAA-PEG solution to a final pH of 1.2 was done with stirring. The pH and transmittance (T) were recorded at 60 s after each titration of HCl. 2.3. Determining Dye Sequestration into Coacervates. Stock solutions of PAA (40 × 10−3 M), F127 (40 × 10−3 M), and PEG (40 × 10−3 M) were prepared. PAA and F127 solutions or PAA and PEG solutions were mixed at equal volumes, together with the dye, MB, JGB, BtB, or ANS separately, to have a final concentration of 20 × 10−3 M PAA, 20 × 10−3 M F127 or PEG, and 0.01 × 10−3 M dye. Addition of 1 M HCl into the prepared solution was done with stirring to form coacervates with sequestered dyes. After 24 h stirring, samples

I(q) = B

⎛ A⎞ 2 2 2 ⎜ ⎟Δρ V P (q) + B ⎝V ⎠

(2) DOI: 10.1021/acs.macromol.6b02815 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules where Δρ is the contrast factor (scattering length density difference between the particle and the solvent), V is the particle volume, A is a prefactor that depends on the number of particles, B is the incoherent background, and P(q) is the form factor for a monodisperse spherical particle with uniform scattering length density (eq 3).28 P(q) =

an abrupt increase in turbidity. The decrease of turbidity at pH 1.8 for the PAA-F127 system indicates the onset of precipitate formation.30 Similarly, from the turbidity measurement of PAAPEG system with HCl titration, one can see the formation of PAA-PEG coacervate starts at pH 3.3, and precipitate formation starts at pH 2.2. It is known that the order of mixing can influence the complexation process, and the evolution of coacervate and precipitate formation might be different under different conditions from those reported here.31,32 3.1. Hydrodynamic Diameter. The hydrodynamic diameter of both PAA-F127 and PAA-PEG coacervates was determined using dynamic light scattering over a range of pH values as shown in Figure S2. The size distribution of 20 × 10−3 M PAA aqueous solution at its natural pH was determined using dynamic light scattering as well, as shown in Figure S3. With the reduction of pH, the hydrodynamic diameter of PAAF127 and PAA-PEG coacervate increases from approximately 60 to 800 nm and 150 to 300 nm separately measured immediately after the formation of coacervation, due to the increase in protonation degree of carboxylate anion functional group of PAA. It has also been reported that the decrease of pH can increase hydrophobicity of PAA-PEO coacervates, and this effect may be in play here, increasing aggregation as pH is reduced.19 In addition, from Figure S2 one can see that the hydrodynamic diameter of the coacervate particles increases with time and reaches stable values after 24 h, holding constant for at least another 24 h, indicating perhaps that surface charge of the droplets is preventing or at least slowing further coalescence. 3.2. Protonation Degree of PAA. FTIR spectra of PAAF127 and PAA-PEG coacervates formed at pH 2.75, 3.00, and 3.25 are shown in Figure S4. The FTIR spectra of PAA-F127 coacervate formed at these pH values indicates the presence of each component, both PAA and F127, within the coacervate. Though the pH range studied here is not able to render 100% protonation of PAA, the carboxyl group is mostly in its protonated state with a peak at ∼1710 cm−1, while the peak representing the ionized carboxylate group at ∼1570 cm−1 is absent for both pure PAA sample prepared at pH 3.25 and PAA-F127 coacervate formed even at the highest studied pH 3.25, indicating that the carboxyl groups of PAA in the PAAF127 coacervate are almost fully protonated in this study. The FTIR spectra of PAA-PEG coacervate prepared at pH values ranging from 2.75 to 3.25 demonstrate the presence of both PAA and PEG in the coacervate as well. FTIR indicates that the carboxylic acid groups of PAA in the PAA-PEG coacervate were mostly protonated (1710 cm−1) at the studied pH, without any carboxylate anion peak (1570 cm−1) visible. The peak at 1645 cm−1 in both PAA-F127 and PAA-PEG coacervates is related to the contained water in the coacervate. On the basis of the FTIR spectra, it is apparent that the protonation degree of carboxyl group of PAA in both PAA-F127 and PAA-PEG coacervate reaches nearly 100% in the studied pH range. Our previous study on the sequestration of methylene blue into polyelectrolyte coacervates reveals that electrostatic interaction does not play a significant role in the sequestration of cationic dye methylene blue, especially at low zeta potential values.4 Zeta potential measurement of PAA-PEG coacervate was carried out as a function of pH, as shown in Figure S5. The zeta potential of PAA-PEG coacervate varies from approximately −12 to −3 mV as the pH drops from 3.25 to 2.59, indicating that the decrease in pH would promote the protonation of PAA. However, it is to be noted that even at

3(sin(qr ) − qr cos(qr )) (qr )3

(3)

where r is the radius of the particle. 2.9. Isothermal Titration Calorimetry (ITC). Isothermal titration calorimetry experiments were performed on a Nano ITC standard volume (TA Instruments). Titration experiment involved 25 injections (10 μL) of 80 mM PAA at 400 s intervals into the sample cell (volume = 1 mL) containing 10 mM PEG solution. Both titrant and titrate had same pH value and KCl concentration. The reference cell was filled with DI water; the experimental temperature and stirring rate was fixed on 25 °C and 250 rpm, respectively. The heat of dilution of 80 mM PAA solution containing different KCl concentrations was later subtracted from the titration data.

3. RESULTS AND DISCUSSION Utilizing the PEO block of F127 as a hydrogen bond acceptor and PAA as a donor, PAA-F127 coacervate was formed under acidic conditions (pH ≤ 3.25) to ensure nearly complete protonation of PAA (pKa ≈ 6.5).29 Similarly, PEG is able to form a hydrogen-bonded complex coacervate at low pH values (pH ≤ 3.25) with PAA as well. For F127, the hydrophilic poly(ethylene oxide) segments stabilize its micellar structure in water and is able to form a hydrogen-bonding network with PAA, while the hydrophobic poly(propylene oxide) block creates a hydrophobic environment within the micelle core. The structures of F127, PEG, and PAA are shown in Scheme 1. The strategy used in this work of coacervate formation with dye sequestration is outlined in Scheme 1. Scheme 1. Schematic Representation of Hydrogen-Bonding Coacervate Formation of PAA and Block Copolymer F127 or PAA and PEG for Sequestration of Dyes from Aqueous Solutiona

a

C* represents critical micelle concentration of F127.

Turbidity was used to qualitatively measure the complex coacervate formation as a function of pH and therefore the protonation degree of carboxylate anion functional group on PAA. Turbidity measurements were performed by lowering the pH with 1 M HCl. Recorded turbidity as a function of pH are shown in Figure S1 (Supporting Information). The PAA-F127 system begins to form coacervate at pH approximately 3.5 with C

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the point at which the ionization of PAA prevents formation of significant amounts of coacervate. The F127 containing coacervate is consistently more efficient at sequestering MB over the pH range of 2.25−3.25, but the difference in the two systems for MB sequestration is not high in absolute terms. For the JGB dye, roughly 80% to more than 90% is captured by the two coacervate systems, depending on pH. Again, the F127 containing system is able to sequester more of the dye. The variation of sequestration of JGB by the F127-containing system as a function of pH is small. This variance is somewhat larger for the PEG containing system, again with a minimum at pH 3.25. The two other dyes (BtB and ANS) are not favorably partitioned into either of the coacervate phases; as little as ∼15% of BtB and never more than ∼45% of BtB is transferred to the coacervate, and for the ANS this range is about 15−30% depending on pH and which system is in question. Again for these dyes, the F127-containing systems are somewhat more effective at capturing the dyes, this difference being large for the pH values of 2.25, 2.5, and 2.75. This sequestration result is somewhat counterintuitive, as one might expect the coacervate’s environment to be more hydrophobic than the waterrich phase, and subsequently a more favorable environment for the dye, but this low amount of partitioning has been previously shown in electrostatic systems where there is no specific interaction between the dye and polyelectrolytes other than electrostatic interactions.4 However, one should point out that the coacervate phase is a relatively small percentage of the overall system’s volume, meaning that these dyes are concentrated into the coacervate phase although not to the same degree as the previously mentioned dyes. To better illustrate the relative uptake ability of coacervate phase for these solutes, partition coefficient was determined. The partition coefficient K can be defined as the ratio of concentration of solute in the polymer-rich phase to the concentration of solute in the water-rich phase, as shown in eq 4. The sequestration and partition coefficient of MB and BtB in PAA-F127 coacervate phase and supernatant are shown in Figures S7a and 7b, respectively. Though the sequestration of BtB into PAA-F127 is only approximately 15−45%, the partition coefficient of BtB is still considerable, varying from roughly 30 to 150 with the variation of pH, indicating that compared to the water-rich phase, BtB prefers the polymer-rich phase. For MB with a sequestration of approximately 70−80%, the partition coefficient is significantly higher than that of BtB, varying from roughly 550 to 920 in the studied pH condition. When the sequestration percentage and the partition coefficients are plotted together, it can be seen that both these two vary with pH in a similar manner. In addition to the study of dye sequestration, furosemide, which is a pharmaceutical compound used to treat hypertension and edema, was selected to show the capacity of coacervate to load pharmaceutical compounds. The partition coefficient of furosemide into PAA-F127 coacervate is shown in Figure S8. The partition coefficients for furosemide into PAA-F127 coacervate and PAA-PEG coacervate are 200−300 and 100− 200, respectively, indicating that the presented hydrogen bond coacervate system here are capable to load furosemide effectively.

the highest pH (3.25) studied in this system, the absolute value of zeta potential of the coacervate is still not high, indicating that even for pH ∼ 3.25, the electrostatic interaction between coacervate and ionic dyes that will be studied later should not be significant. 3.3. Sequestration of Dyes into Coacervates. In order to study the sequestration process of dyes into the hydrogenbonded coacervate, PAA-F127 and PAA-PEG coacervate samples over a range of pH (2.25−3.25) were made. The dyes used include two cationic dyes (MB and JGB), an anionic dye (ANS), and a nonionic dye (BtB). Solutions were prepared with each polymer and a dye, and then the solutions of hydrogen bond donor and acceptor polymers were mixed together. The final polymer concentrations were 20 × 10−3 M of each polymer with respect to its functional group, which is lower than CMC of F127, and 0.01 × 10−3 M of the dye. Solutions were also made with concentrations of 200 × 10−3 M in each polymer, which is higher than CMC of F127, and 0.1 × 10−3 M of the dye. The mixtures were initially made at a higher pH value where the coacervate does not form. This process was selected to ensure good mixing between polymers and dyes. The pH of the mixture containing both polymers and the dye was then adjusted to form a complex coacervate with the sequestered dye. As shown in Figure S6, over the pH range from 2.25 to 3.25, sequestration of MB into both PAA-F127 and PAA-PEG coacervates reaches an equilibrium after 24 h stirring and remains constant over the next 24 h. Figure 1 shows the sequestration of the four different dyes, namely MB, JGB, BtB, and ANS, into PAA-F127 and PAA-PEG

Figure 1. Sequestration of (a) MB, (b) JGB, (c) BtB, and (d) ANS into PAA-F127 and PAA-PEG coacervates at different pH. The initial dye concentration in all solutions was 0.01 × 10−3 M. Coacervates were prepared using 20 × 10−3 M PAA and 20 × 10−3 M F127 or 20 × 10−3 M PAA and 20 × 10−3 M PEG with the existence of dyes by adjusting pH.

coacervates formed at different pH values. Sequestration of dye into the coacervate depends strongly on the chemical structure of the dye. Two of the dyes, MB and JGB, are sequestered at a relatively high proportion into the two complex coacervates. Amounts ranging from 70 to 80% of the MB dye are sequestered into the two types of coacervate, depending on pH, with maximum sequestration occurring at pH 3.0. Sequestration is lowest at pH 3.25, presumably as this is near

K= D

[solute in polymer‐rich phase] [solute in water‐rich phase]

(4)

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Macromolecules 3.4. Influence of Hydrogen-Bonding Formation on Dye Sequestration. To understand the reason for the large difference between sequestration of MB and JGB and the sequestration of BtB and ANS into the PAA-F127 and PAAPEG coacervates as well as to elucidate more about the interaction between these dyes with the coacervates, UV−vis spectroscopy was used to examine the dyes in both aqueous solution and within the coacervate phases. Figure 3a,b and Figure S9 show the UV−vis spectra of MB, JGB, BtB, and ANS in aqueous solutions containing just one polymer. The UV−vis spectra of MB, JGB, BtB, and ANS in PAA-F127 or PAA-PEG coacervate phases compared with that in aqueous solution without any addition of polymer are shown in Figure 2. From

668 nm (Figure 3a). The same red-shift exists in the PAAF127/MB and PAA-PEG/MB complex coacervates as well (Figure 2a). This shift is characteristic of the formation of hydrogen bonds.33,34 JGB in aqueous solution alone shows a λmax of 615 nm. Similarly, with the addition of F127 or PEG, the λmax of JGB still remains at 615 nm, while with PAA added, the λmax of JGB red-shifts from 615 to 628 nm (Figure 3b). Figure 2b shows the existence of the same red-shift in the PAAF127/JGB and PAA-PEG/JGB coacervates as well. The redshifts of the MB and JGB spectra indicate the formation of hydrogen bonds between MB or JGB and PAA. It is to be noted that the observed λmax shift of MB and JGB in the presence of PAA is a combination of the λmax of free dye and of the hydrogen-bonded dye interacting with PAA, since the hydrogen-bonding complex is in equilibrium with free dye. UV−vis measurement is a commonly used method to determine the association constants for supramolecular systems whit a maximum absorbance wavelength shift upon association, using Beer−Lambert’s law.35 UV−vis experiments wherein the concentration of dye is kept at 0.01 × 10−3 M in aqueous solution while varying the concentration of PAA, which is always kept in excess, were also carried out to determine the equilibrium association constants (Ka) for the hydrogenbonded complex formation between dye and PAA. Each of the absorbance values for MB or JGB with a varying concentration of PAA in aqueous solution is treated as the sum of the absorbance contribution from free dye and hydrogen-bonded dye interacting with PAA according to the eq 5, where A is the absorbance and i indicates the specific wavelength. Using the Beer−Lambert law, this equation may be rewritten as eq 6. Since b is the path length, it is assumed to be the same for both the free and hydrogen-bonded dye. Using eq 6, one can calculate the concentration of free dye and the dye involved in hydrogen-bonding complex formation. If we assume that the hydrogen-bonding complexation between dye and PAA is a bimolecular equilibrium process as shown in eq 7, eq 8 can be used to express the equilibrium association constant, which may be rewritten as a linear relationship using logarithm transformation, as shown in eq 9. A plot of ln{[dye]hb/ [dye]free} vs ln[−COOH]free as shown in Figure 3c yielded two straight lines in the form of ln{[dye]hb/[dye]free} = 1.25 ln[−COOH]free − 3.02 (R2 = 0.9531) for MB and ln{[dye]hb/ [dye]free} = 1.29 ln[−COOH]free − 1.18 (R2 = 0.9905) for JGB. The equilibrium association constant for the hydrogen bond formation between dye and −COOH groups can be calculated from the fitted intercept using eq 6, yielding a Ka of 0.049 for MB and 0.31 for JGB, which again illustrates the higher sequestration of JGB into coacervates than that of MB since the

Figure 2. UV−vis curves of (a) MB in aqueous solution, PAA-F127/ MB and PAA-PEG/MB coacervates, (b) JGB in aqueous solution, PAA-F127/JGB and PAA-PEG/JGB coacervates, (c) BtB in aqueous solution, PAA-F127/BtB and PAA-PEG/BtB coacervates, and (d) ANS in aqueous solution, PAA-F127/ANS and PAA-PEG/ANS coacervates. All the coacervates were prepared using 20 × 10−3 M PAA and 20 × 10−3 M F127 or 20 × 10−3 M PAA and 20 × 10−3 M PEG with the addition of 0.01 × 10−3 M MB, 0.01 × 10−3 M JGB, 0.01 × 10−3 M BtB or 0.02 × 10−3 M ANS at pH 2.75. The pH of aqueous solutions with dyes was adjusted to 2.75 as well.

this data, the presence of hydrogen bonds between MB and PAA can be validated. In the absence of any polymer, MB in aqueous solution exhibits a maximum absorbance wavelength (λmax) of 664 nm. With the increasing addition of F127 or PEG, the λmax of MB is still 664 nm (Figure S9), while with the addition of PAA, the λmax of MB exhibits a red-shift from 664 to

Figure 3. UV−vis curves of 0.01 × 10−3 M (a) MB and (b) JGB with the addition of increasing concentration of PAA. The red-shift in maximum absorbance wavelength indicates the intermolecular interaction between PAA and the studied dyes. (c) Linear plot of ln{[dye]hb/[dye]free} vs ln[−COOH]free, in the form of ln{[dye]hb/[dye]free} = 1.25 ln[−COOH]free − 3.02 (R2 = 0.9531) for MB and ln{[dye]hb/[dye]free} = 1.29 ln[−COOH]free − 1.18 (R2 = 0.9905) for JGB. E

DOI: 10.1021/acs.macromol.6b02815 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

PEG coacervate, indicating that the microenvironment of PAAF127 coacervate is more nonpolar than that of PAA-PEG coacervate. Looking at the structure of F127 and PEG, F127 consists of hydrophilic polyoxyethylene units (70%) and hydrophobic polyoxypropylene blocks (30%), while PEG comprises of hydrophilic polyoxyethylene units only. The structure of F127 enables it to form micelle with a polyoxyethylene shell which can interact with PAA through hydrogen bonds and a poly(propylene oxide) core which creates a more hydrophobic environment within the micelle. Therefore, the PAA-F127 coacervate is more hydrophobic than PAA-PEG coacervate, which is in consistent with the fluorescence spectra of PAA-F127/ANS and PAA-PEG/ANS coacervates. The higher hydrophobicity of the PAA-F127 coacervate compared to that of the PAA-PEG coacervate ensures a better sequestration capacity of PAA-F127 coacervate for all the four dyes. In order to further confirm the role of hydrophobicity in the partitioning process of dyes into coacervates, the sequestration of MB and BtB was studied with poly(methacrylic acid) (PMAA)-F127 and PMAA-PEG coacervates as well, shown in Figure S10. MB is an example of a dye which hydrogen bonds with the polyacid, and BtB is an example of dye for which hydrophobicity is the driving force for sequestration. The increased hydrophobicity of PMAA in water from the α-methyl group compared to that of PAA has been investigated in many publications.38,39 As shown in Figure S10, the difference of MB sequestration between PAA-F127 or -PEG and PMAA-F127 or PEG coacervate systems is almost negligible in absolute terms, which is not like the sequestration of MB into PAA-F127 and PAA-PEG coacervate, where the increased hydrophobicity by F127 promotes the uptake of MB. To better understand this phenomenon, one may need to take the effect from both hydrogen bond and hydrophobicity on the sequestration of MB into consideration. Though replacing PAA with PMAA would probably increase the hydrophobicity within coacervate, the αmethyl group of PMAA would probably inhibit the hydrogen bond formation between carboxylic acid group and MB due to the steric hindrance.40−42 Therefore, because of the cocontribution of reduced probability for hydrogen bond formation and increased hydrophobicity within coacervate, the difference of sequestration of MB into PAA- and PMAA-containing coacervate is negligible. However, using the PMAA-F127 or -PEG coacervate system, sequestration of BtB is increased by ∼10% compared with PAA-F127 or -PEG coacervates, indicating that the enhancement of hydrophobicity promotes the sequestration of dyes with hydrophobic interaction alone as the driving force. 3.6. Influence of Added Salt on Coacervate Formation. It was observed that the addition of salts such as NaCl and KCl was able to shift the pH window over which coacervate is formed. In order to better understand this phenomenon, potentiometric titrations of PAA was performed with the addition of either NaCl or KCl with concentrations ranging from 0 to 500 mM, as shown in Figure S12. Figure S13 shows that as the concentration of added salt increases from 0 to 500 mM, the pK a of PAA obtained from these potentiometric titration curves drops from 6.5 to 5.0, indicating that in order to attain the same protonation degree of PAA, a lower value of solution pH is needed for higher salt concentration. A similar influence on pKa value is observed for both salts. The turbidity of PAA-F127 and PAA-PEG systems with a range of added NaCl and KCl concentrations

equilibrium association constant for JGB is higher than that of MB with PAA. One possible explanation for the deviation of the slope of these lines from 1 to roughly 1.3 is that the carboxylic acid functional groups of the PAA may be interacting with themselves to form dimers or hydrogen bonding with water molecules, as carboxylic acid groups are known to do.36 These interactions were not accounted for in this analysis. Ai = Ai (free dye) + Ai (hydrogen‐bonded dye)

(5)

Ai = εi(free dye) × b × c(free dye) + εi(hydrogen‐bonded dye) × b × c(hydrogen‐bonded dye)

−COOH + dye ↔ −COOH···dye Ka =

ln

[dye]hb [−COOH···dye] = [−COOH][dye] [−COOH]free [dye]free

[dye]hb = ln[−COOH]free + ln K a [dye]free

(6) (7)

(8)

(9)

In contrast, as shown in Figure 2c,d and Figure S9, the λmax of both BtB and ANS remains fixed at 430 and 351 nm separately either with the addition of polymers into the aqueous solution or in the hydrogen-bonded coacervates, indicating that there is no hydrogen bond formation between BtB and polymers or ANS and polymers. The primary mechanism for sequestration of BtB and ANS into either PAA-F127 or PAAPEG coacervates is hydrophobic interactions, while for the sequestration of MB and JGB into PAA-F127 as well as PAAPEG coacervates, the co-contributions of both hydrophobic interactions and hydrogen bonds lead to a significantly higher sequestration capacity over the studied pH range compared to the sequestration of BtB and ANS. 3.5. Influence of Coacervate Hydrophobicity on Dye Sequestration. Over the pH ranges where PAA-F127 and PAA-PEG coacervate formation is observed, PAA-F127 coacervate system always shows a higher sequestration than PAA-PEG coacervate system for all these four dyes. In order to better understand this phenomenon, we used fluorescence spectroscopy and ANS as a probe to investigate the physicochemical environment within the coacervates. Previous studies have shown that the photophysical properties of ANS are highly sensitive to polarity of the environment.37 Figures 6a and 6f show the fluorescence spectra obtained from PAA-F127/ ANS and PAA-PEG/ANS coacervates prepared using 20 × 10−3 M PAA and 20 × 10−3 M F127 or 20 × 10−3 M PAA and 20 × 10−3 M PEG with the addition of 0.1 × 10−3 M ANS at pH 3.25. Specifically, the fluorescence spectra for PAA-F127/ ANS and PAA-PEG/ANS coacervates showed a broad feature between 400 and 650 nm, which indicates a combination of different microenvironments of varying polarities. Deconvolution of the spectra by Gaussian fitting provides two specific emission maxima associated with two distinct microenvironments for ANS. Specifically, both the fluorescence spectra for PAA-F127/ANS and PAA-PEG/ANS coacervates displayed an emission maximum at 470 nm and another peak at 530 nm (Figure 6). The former peak was assigned to the nonpolar (more hydrophobic) excited state localized on the naphthalene ring of ANS, while the latter was consistent with the emission from the charge transfer state (a more hydrophilic environment). The relative intensity of the 470 nm peak of sequestered ANS into PAA-F127 coacervate is larger than that for PAAF

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Macromolecules was studied as a function of pH, shown in Figure S11. The turbidity data show that as the concentration of added NaCl or KCl increases, pHφ, where the formation of coacervate starts, shift to higher pH values, indicating that for PAA fewer carboxylate groups need to be protonated to form coacervate with F127 or PEG. It can be therefore said that the existence of salt in the solution promotes coacervate formation between PAA and F127 or PEG. Again, this is a somewhat counterintuitive result, given that at those higher pH values a larger percentage of the PAA carboxylic acid groups will be charged. However, it is also known that PEG can form crown ether like complexes with metal ions such as sodium and potassium, creating charged complexes.43,44 It has also been suggested that the addition of inorganic salt will deteriorate the thermodynamic quality of the solvent with respect to weakly complexing polymers such as PEG, resulting in the increased pHφ with the addition of salt.19 Zeta potential of PAA-PEG coacervate formed at pH 3.25 with increasing addition of KCl is shown in Figure S14. It has been mentioned before that the presence of KCl in aqueous solution would promote the deprotonation of PAA, leading to a higher charge density of PAA at a given pH. Despite the fact that addition of KCl would increase ionization degree of PAA at the given pH, the zeta potential of PAA-PEG coacervate tends to be less negative as the KCl concentration increases, indicating that potassium ion likely interacts with PEG to form crown ether like, positively charged complexes, which reduces the absolute zeta potential of formed PAA-PEG coacervate with the addition of KCl. Isothermal titration calorimetry (ITC) was used to study the influence of KCl addition on the hydrogen-bonded coacervate formation process by titrating PAA into PEG with the presence of different concentration of KCl in both PAA and PEG solutions at pH 3.2, as shown in Figure 4. Though the

Because of the low charge density in this study, it is reasonable to assume that electrostatic interaction between the incompletely protonated PAA and PEG which forms crown ether like complexes with potassium would have little contribution to the coacervate formation. In this ITC experiment, the assumption that hydrogen bond rather than electrostatic interaction leads to the coacervate formation regardless of KCl concentration studied here will be made. A careful observation of Figure 4 reveals that the interaction between PAA and PEG occurs in two steps, which is similar to the results in polyelectrolyte complexation. The first step of the complexation process describes the formation of soluble complexes via hydrogen bonding interaction between PAA and PEG. The bound PAAPEG chains of the first step aggregates to form a dense, intermolecular, fluctuating polymer network (complex coacervates) in the second step. We observe that the thermodynamic characteristics of both steps are affected by the addition of KCl. More specifically, the overall change in energy (endothermic for first step and exothermic for second step) becomes smaller as the salt concentration is increased, which means that the driving force for complexation weakens. This change can be explained by the complexation of potassium with PEG as well as the slightly reduced protonation degree of PAA upon addition of KCl, limiting the opportunity for PEG to interact with PAA via hydrogen bonding. As mentioned before, it has been suggested that the addition of inorganic salt will deteriorate the thermodynamic quality of the solvent with respect to polymers.19 Therefore, even though the addition of KCl weakens the complexation between PAA and PEG, the deterioration of solvent thermodynamic quality still leads to an increased pHφ with the addition of salt. 3.7. Influence of Added Salt on Sequestration of Dyes. Sequestration of MB and BtB into both PAA-F127 and PAA-PEG coacervates was performed at pH 2.75 with a constant dye concentration of 0.01 × 10−3 M under a range of added NaCl and KCl concentrations, shown in Figure 5. The sequestration of MB into PAA-F127 coacervate drops from approximately 80% to 50% as the concentration of added salt

Figure 4. ITC analysis describing the formation of the complex coacervate through interaction of PAA and PEG in the presence of KCl with different concentrations in both titrate and titrant. 80 mM PAA solution with known KCl concentration was titrated into 10 mM PEG solution with the same solution quality, pH, and KCl concentration. The background was obtained from the titration of titrant into corresponding solvent that was later subtracted to exclude the heat of dilution.

ionization degree of PAA would be increased by the presence of KCl, the FTIR spectrum as shown in Figure S4 of PAA-PEG coacervate formed at pH 3.2 with presence of 200 mM KCl shows no obvious peak at ∼1570 cm−1 attributed to deprotonated carboxylate group, indicating the low charge density of PAA under the studied condition. Voorn and Overbeek suggested that coacervation can take place only beyond a critical charge density and/or chain length.45,46

Figure 5. Sequestration of MB (a) with the addition of NaCl, (b) with the addition of KCl using PAA-F127 and PAA-PEG coacervates, and of BtB (c) with the addition of NaCl, and (d) with the addition of KCl using PAA-F127 and PAA-PEG coacervates All the coacervates were prepared using 20 × 10−3 M PAA and 20 × 10−3 M F127 or 20 × 10−3 M PAA and 20 × 10−3 M PEG with the addition of 0.01 × 10−3 M MB or 0.01 × 10−3 M BtB at pH 2.75. G

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Figure 6. Fluorescence emission spectra of PAA-F127/ANS coacervate dispersions with the addition of (a) 0 mM, (b) 20 mM, (c) 50 mM, (d) 100 mM, (e) 200 mM NaCl and PAA-PEG/ANS coacervate dispersions with the addition of (f) 0 mM, (g) 20 mM, (h) 50 mM, (i) 100 mM, and (j) 200 mM NaCl recorded at room temperature. The coacervates were prepared using 20 × 10−3 M PAA and 20 × 10−3 M F127 or 20 × 10−3 M PAA and 20 × 10−3 M PEG with the addition of 0.1 × 10−3 M ANS and different concentration of NaCl at pH 3.25. The coacervate dispersions were excited at 350 nm, and emission spectra were recorded from 400 to 650 nm. Multi-Gaussian fitting was used to deconvolute the fluorescence emission spectra of PAA-F127/ANS and PAA-PEG/ANS coacervates. Sequestration of BtB into and I470/I530 of ANS within (k) PAA-F127 and (l) PAAPEG coacervates as a function of added NaCl.

NaCl. The fluorescence spectra of ANS within the PAA-F127 and PAA-PEG coacervates with a range concentration of NaCl were obtained. Deconvolution of the spectra by Gaussian fitting provides two specific emission maxima associated with two distinct microenvironments for ANS, specifically, one emission maximum at approximately 470 nm and another peak at approximately 530 nm. The former peak is related to the nonpolar environment or hydrophobicity in the coacervate, while the latter is characteristic of the polar environment in the coacervate.47 Here, the intensity ratio of the former (470 nm) to the later (530 nm) peak (I470/I530) is used to quantify the hydrophobicity of the coacervate. The plot of BtB sequestration using PAA-F127 and PAA-PEG coacervate as well as I470/I530 with the addition of NaCl is shown in Figures 6k and 6l; both the sequestration of BtB and the hydrophobicity of PAA-F127 and PAA-PEG coacervates increase with the addition of salt. This consistency trend of BtB sequestration and I470/I530 with the increasing concentration of added NaCl indicates that hydrophobicity is crucial for the sequestration of BtB into the coacervates. 3.8. Influence of Micelle Formation on the Sequestration of Dyes. Block copolymers with amphiphilic character, including Pluronic copolymers, are known to assemble in aqueous solution to form micelles with a core−shell architecture when the concentration reaches its critical micelle concentration, where hydrophobic segments are segregated from aqueous exterior to form an inner core surrounded by a crown of hydrophilic segments. These block copolymer micelles have been used as drug carrier systems because of the high drug-loading capacity of the inner core resulting from

increases from 0 to 500 mM. Similarly, for PAA-PEG coacervate, the sequestration of MB decreases from approximately 70% to 45% with the increase of either NaCl or KCl concentration from 0 to 500 mM. However, for BtB, the trend of sequestration with increasing concentration of NaCl or KCl is completely opposite to that of MB. With the addition of either NaCl or KCl whose concentration ranges from 0 to 500 mM, the sequestration of BtB increases from 30% to 80% for PAA-F127 coacervate and 20% to 50% for PAA-PEG coacervate separately. For the sequestration of MB into PAA-F127 and PAA-PEG coacervates, the main driving force is hydrogen bonding interaction rather than hydrophobic interaction, which can explain the reduction of MB sequestration with the increasing concentration of added salt at a given pH, since the pKa of PAA drops from 6.5 to 5.0 as the concentration of added NaCl or KCl increases from 0 to 500 mM, as shown in Figure S13. This drop of pKa indicates that at a given pH the protonation degree of PAA decreases with the addition of salt. At pH 2.75, the number of carboxylic acid groups which are able to form hydrogen bond with MB is lowered by the by the addition of salt, therefore inhibiting the sequestration of MB via hydrogenbonding interaction. However, the main mechanism for the sequestration of BtB is hydrophobic interaction since there is no hydrogen-bonding interaction between BtB and PAA-F127 or PAA-PEG coacervates. In order to obtain a better explanation for the increasing sequestration of BtB with the addition of NaCl or KCl, ANS was used as a probe to study the hydrophobicity of PAA-F127 and PAA-PEG coacervates with the addition of H

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Figure 7. Normalized intensity autocorrelation function C(τ) plotted for (a) a pure F127 aqueous solution with a concentration of 20 × 10−3 M and 200 × 10−3 M and (b) a pure P84 and P123 aqueous solution with a concentration of 20 × 10−3 M. The insets in (a, b) provide the associated size distribution from these autocorrelation functions. (c) Small-angle X-ray scattering patterns for aqueous solutions and the F127-PAA coacervate suspension for a variety of polymer concentrations. The dashed line for the 200 mM PAA-Fl127 coacervate is a fit to the form factor of a sphere. These scattering patterns are consistent with the schematic morphologies: (1) High concentration of micelles in the coacervate (200 mM PAAF127), (2) a concentrated polymer complex in solution with limited concentration of micelles (20 mM PAA-F127 coacervate), (3) micellar solution, and (4) unimers. At low concentration of micelles, it is difficult to distinguish between (3) and (4).

this low concentration solution even though this is known to be greater than the CMC from F127.51 However, this scattering profile is consistent with the scattering from dilute (1 wt %) Pluronic P85 micelles.27 Additionally when more carefully examining the scattering near q = 0.025 Å−1, the curvature in the scattered intensity is slightly downward as is observed for scattering from a common length scale as opposed to the upturn in intensity from larger scale scatterers. As the scattering intensity, I(q), is proportional to the number density of scatterers,28 the shoulder in the scattering associated with the form factor of the sphere is only well resolved at higher concentrations.27 As the unimers and micelles are in dynamic equilibrium, concentrations much greater than the CMC are required to obtain a large density of micelles. This is particularly relavent for Pluronic surfactants where the unimer-to-micelle transition is broad and evolves over a decade in concentration.51 Nonetheless, the emergence of a shoulder in the scattering profile provides some evidence for micelles.52 The scattering from the coacervates is in general more intense, which can be associated partially with the concentrating of the polymers within the coacervate phase. At low concentration (20 × 10−3 M PAA and F127), the scattering profile exhibits a strong upturn at low q, which is associated with the large scale heterogeneities in the coacervate. The large scale density fluctuations are consistent with the opaque appearance of the coacervate from light scattering (based on the large difference in the refractive index between water and the polymers). Moreover, scattering patterns from PAA/ poly(N,N- dimethylaminoethyl methacrylate) coacervates53 and gelatin/polyelectrolyte complexes54 are very similar qualitatively to the scattering obtained from the PAA-F127 coacervate at low concentrations. This similarity indicates that the scattering is dominated by the concentration fluctuations in polymer−water, not micelles as these other systems do not provide a mechanism for nearly monodisperse micelles. However, as the scattering is statistical, we can only infer that the concentration of micelles, if any, is small in this coacervate.

its hydrophobicity. A question that arises is whether the formation of micelle will have any enhancement in the hydrophobicity of the coacervate phase and therefore the dye sequestration. To demonstrate this, PAA and F127 with a concentration of 20 × 10−3 M PAA and 20 × 10−3 M F127 below the CMC of F127 as well as 200 × 10−3 M PAA and 200 × 10−3 M F127 over the CMC of F127 were used to prepare the corresponding coacervates. DLS measurements of 20 × 10−3 M and 200 × 10−3 M F127 were performed at room temperature to investigate the micelle formation with the concentration below or over CMC by measuring the normalized intensity autocorrelation functions C(τ), as shown in Figure 7a. As shown in the inset of Figure 7a, the hydrodynamic diameters of F127 obtained from the autocorrelation functions with a concentration below CMC and over CMC are approximately 8.3 and 21.4 nm, respectively, which is consistent with the values reported in previous literature,48,49 indicating that F127 exists in form of unimer at concentration of 20 × 10−3 M while it forms micelle at concentration of 200 × 10−3 M. In order to better understand the internal structure of the coacervates, small-angle X-ray scattering (SAXS) was employed as shown in Figure 7c. The SAXS patterns for the individual components at the concentrations used to form the coacervate show almost no features. For the lowest concentration of F127 (20 × 10−3 M), the scattering intensity, I(q), was nearly invariant across the q range examined, except for a very weak upturn at low q. This scattering profile is consistent with individual F127 chains in solution (unimers). The slight upturn in I(q) at low q is likely due to the large scale density fluctuation from the polymer clustering.50 This upturn becomes more pronounced at higher concentration (200 × 10−3 M) of F127. This could be a result of the convolution of the shoulder from the form factor of spheres and the low q upturn associated with large scale density fluctuations (same as at lower concentration of F127). From the scattering profiles alone, it is difficult to definitively determine that micelles are present in I

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Macromolecules This might be expected as the F127 concentration was below the CMC in the solutions used. However, the concentration of F127 locally in the coacervate is larger than the total concentration, so this effective concentrating of the surfactant could increase the number density of micelles relative to their initial concentration. For the higher concentration coacervate (200 × 10−3 M PAA and F127), there is a dramatic shift in the scattering shape. The profile is sigmoidal with a shoulder-like feature at low q. This scattering profile is consistent with the SAXS profiles of Pluronic micelles at much higher concentrations, but still below the nematic crystal transitions.52 As shown by the dashed line in Figure 7c, this scattering profile can be well described by the form factor, P(q), of a monodisperse hard sphere. From this fit, the average radius of the micelles within the coacervate droplets is approximately 90 Å. This size scale is consistent with the radius of F127 micelle reported by Kadam et al.55 and the hydrodynamic radius of F127 micelle reported by Pragatheeswaran et al.56 Examining the scattering from the pure F127 and the coacervate at the same concentration (200 × 10−3 M) reveals significant differences in the profile. The low q upturn associated with the concentration fluctuations appears to dominate the scattering for the pure F127, while the form factor for the micelles dominates in the scattering for the coacervate. This suggests that the number density of micelles is increased in the coacervate. There are several reasons why the density of micelles might increase in the coacervate. First, the local concentration of F127 is increased in the coacervate so the equilibrium between unimers and micelles is shifted toward additional micelles. Second, the interactions between the PAA and the ether oxygen in PEO can significantly alter the thermodynamics in these systems. Their hydrogen bonding has been suggested to promote micellization in aqueous solution,56 while the addition of PAA to neat F127 in the solid state leads to a disorder-to-order transition.57 Additionally, there has been some suggestion that van der Waals forces and hydrophobic interactions are also important factors in the enhanced aggregation of F127 micelles in PAA.58 Based on these prior works relating to the micelle formation of F127 in the presence of PAA, it is not surprising that the coacervates exhibit a higher density of micelles that lead to the observed scattering patterns in Figure 7c. The sequestration of MB and BtB into PAA-F127 coacervates prepared using a mixture of PAA and F127 solution with a concentration at either 20 × 10−3 or 200 × 10−3 M was studied. As shown in Figures 8a and 8b, whether the F127 forms micelle or not does not have a significant influence on the sequestration of MB and BtB into PAA-F127 coacervates. As shown in previous study, hydrogen bonding is the main driving force for the uptake of MB into PAA-F127 coacervates; therefore, it is not surprising that the F127 micelle formation does not affect the sequestration of MB. However, to our surprise, the F127 micelle formation does not have any obvious increase in the sequestration of BtB even the previous study showed that hydrophobic interaction is the main driving force for BtB uptake into coacervates. Figures 8c and 8d show the sequestration of MB and BtB into PAA-F127, PAA-P84 and PAA-P123 coacervates prepared using 20 × 10−3 or 200 × 10−3 M PAA and Pluronic polymer. Similarly, using different Pluronic block copolymer does not have a significant influence in the sequestration of MB. However, for the sequestration of BtB, using hydrophobic interaction as the main driving force for the uptake, one can see that the sequestration varies a lot with

Figure 8. Sequestration of (a) MB and (b) BtB into PAA-F127 coacervates formed using 20 × 10−3 M PAA and 20 × 10−3 M F127 or 200 × 10−3 M PAA and 200 × 10−3 M F127. Sequestration of (c) MB and (d) BtB into PAA-F127, PAA-P84, and PAA-P123 coacervates prepared using 20 × 10−3 M PAA and 20 × 10−3 M pluronic block copolymers including F127, P84, and P123.

the selection of different Pluronic polymers. As shown in Figure 7b, obviously with this low concentration at 20 × 10−3 M, P84 exists in form of unimer while P123 forms micelle in aqueous solution. However, when we plot the sequestration of BtB with PPO wt %, one is able to figure out that the sequestration of BtB increases linearly with the increase of PPO wt % of the used Pluronic block copolymer, regardless of whether the block copolymer forms micelle or not. In other words, these results indicate that the micelle formation does not contribute significantly to the hydrophobicity within the coacervate droplets. 3.9. Release of Dyes Out of Coacervates. A question that arises is whether the small molecule dyes can move easily between the two phases or between coacervate and fresh water. That is to say, once sequestered, will the dyes remain in the coacervate or not? Especially given the fact that the surface tension is low between the two phases, it is plausible to assume that the sequestered material may not remain sequestered. This was probed by removing the supernatant and replacing it with DI water multiple times. In this experiment, the coacervate phase was separated from the water-rich phase using centrifugation, and the dye concentration in the supernatant was determined at the start (denoted as 0 × removal) and then replaced by Milli-Q deionized 18.2 MΩ water, which has had its pH adjusted to be the same as that of the supernatant and has the same volume as the supernatant. After 24 h stirring of the mixture, the dye concentration in the supernatant was determined (denoted as 1 × removal). This procedure was then repeated (denoted as 2 × removal). The results for these removal experiments are shown in Figure 9. For all the four dyes and the two coacervate systems, some dye is seen to diffuse from the coacervate to the fresh Milli-Q water. Quantitatively, one can see that both the PAA-F127 and PAA-PEG coacervate can have significantly higher retention of MB and JGB compared to BtB and ANS. The dyes that are better retained in the coacervate phases are those that are able to interact with the coacervate through both hydrophobic and hydrogen bonding, indicating that hydrogen bonds play a more J

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dyes over BtB and ANS. Previous studies have shown that the photophysical properties of ANS are highly sensitive to polarity of the environment.37,59 Using ANS as a probe, fluorescence spectra of ANS within PAA-F127 and PAA-PEG coacervates was measured to determine the hydrophobicity of coacervates. Our study shows that the higher hydrophobicity of PAA-F127 coacervate over PAA-PEG coacervate ensures a better sequestration capacity of PAA-F127 coacervate for all the four dyes. At a given pH, the sequestration of MB into both PAA-F127 and PAA-PEG coacervates decreases with the addition of salt, while the sequestration of BtB into coacervates increases with the addition of salt. Potentiometric titration curves of PAA with the addition of salt shows that the pKa of PAA drops from 6.5 to 5.0 as the concentration of added NaCl or KCl increases from 0 to 500 mM, indicating that the addition of salt will inhibit the protonation of PAA, which can explain the lowered sequestration of MB with the addition of salt, since the main driving force to sequester MB is hydrogen bonding interaction between MB and PAA. However, the fluorescence spectra of ANS within coacervates reveals that the addition of salt will promote the hydrophobicity within both PAA-F127 and PAAPEG coacervates, which can explain the increased sequestration of BtB with the addition of salt.

Figure 9. MB, JGB, BtB, and ANS concentration in the supernatant during the supernatant removal experiment. The initial feeding concentration of dyes was 0.01 × 10−3 M. During each removal cycle “supernatant” refers to fresh DI water that is added.

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crucial role in the ability to slow diffusion of dye out of the coacervate phase into water.

ASSOCIATED CONTENT

S Supporting Information *

4. CONCLUSION Presented here is a study of the sequestration of four dyes MB, JGB, BtB, and ANSinto hydrogen-bonded coacervates. Hydrogen-bonded coacervates were formed by simply mixing the PAA, F127, or PEG as well as the dye in aqueous solution and adjusting the solution pH. The dual contribution of both hydrogen bonding formation between MB or JGB and PAA and hydrophobicity within the coacervate environment enables a much higher sequestration than that for the other two dyes, BtB and ANS, which are partitioned into coacervates by hydrophobic interaction only. In fact, for the dyes without hydrogen bonding interaction with coacervates, only a minority of BtB and ANS molecules were partitioned into the coacervate phases, showing that the specific interactions are very important and that the hydrophobicity of the coacervate environment in the absence of added salt is not very high. This of course should be understood with the caveat that the coacervate phase is a small percentage of the overall system volume, meaning that in all cases dyes are concentrated into the hydrogen bonding coacervate. However, the addition of NaCl or KCl to the PAAF127 or PAA-PEG coacervate system significantly promotes the hydrophobicity within the coacervate and therefore increases the sequestration efficiency of BtB into coacervate. At the same time, the addition of NaCl and KCl inhibits the protonation of PAA and reduces the number of carboxylic acid groups available for MB to form hydrogen bond, therefore reducing the uptake of MB into both PAA-F127 and PAA-PEG coacervates with increased salt concentration. This work may present insight into the role of intramolecular interactions in the partition of small molecules into coacervates, a better understanding of the environment within the coacervates, and the influence of existent salt on the environment within the coacervates. UV−vis spectra of the dyes either in the aqueous solution with the addition of polymer or in the coacervate state showed hydrogen bond formation between MB and PAA or JGB and PAA, which enables a much higher sequestration of these two

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b02815. Turbidity data, dynamic light scattering data, FTIR, zeta potential data, UV−vis spectra, sequestration and partition coefficient for additional small molecules in the various polymer complex coacervate systems, and potentiometric titration data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (N.S.Z.). ORCID

Chao Wang: 0000-0002-5205-9771 Bryan D. Vogt: 0000-0003-1916-7145 Tianbo Liu: 0000-0002-8181-1790 Nicole S. Zacharia: 0000-0001-7925-1416 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from NSF Award DMR1425187 as well as support from the University of Akron’s NSF REU site DMR-1359321. The authors also thank Prof. Xiong Gong of the University of Akron for use of his UV−vis equipment.

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