Effect of mixed solvents on polyelectrolyte complexes with salt

Strongly interacting polyelectrolyte complexes (PECs) are a versatile class of materials whose physical states can be driven from solids into liquids and ultimately into homogenous solution upon salt addition. However, many of these materials can display high stability using common monovalent salts, leading to difficulties in accessing the entire PEC spectrum. Here, the model system, composed of two styrenic polyelectrolytes, required exceptionally high salt to drive phase transition.We term the amount of salt required to drive these transitions salt resistance. In water, the PEC transferred from solid into liquid at 2.5 M NaBr and never fully dissociated within the studied salt range. We discovered an unconventional approach of weakening salt resistance by switching the solvent to miscible ethylene glycol/water and ethanol/water, allowing us to systematically introduce more hydrophobic constituents. Employing microscopy to determine physical states qualitatively, we found that higher hydrophobicity lowered salt resistance for phase transition and disassembly.

In this mechanism, depending on the chemical and ionic nature of the pair of polyelectrolytes, the physical state of PEC materials can span from glassy solids to low viscosity liquids [19][20][21][22][23]. The phase of the final material has been conventionally hypothesized to be undoubtedly dominated by the nature of polyelectrolyte chain components [18,[24][25][26]. For instance, Li et al. have shown that the self-assembly of two charged hydrophilic polypeptides, poly(lysine) and poly(glutamic acid), resulted in a low viscosity liquid in water [27]. The polypeptides have carbonyl and amino groups along the chain backbone that facilitate hydrogen bonding, and the charged moieties can deprotonate depending on pH conditions. In contrast, the PEC system studied in this present work, composed of poly[(vinylbenzyl) trimethylammonium chloride] (PVBTMA) and poly[sodium 4-styrenesulfonate] (PSS), formed solid precipitates in water (Scheme 1). Here, the aliphatic backbone and aromatic side groups increase the hydrophobicity of the polyelectrolytes, and the trimethylammonium and sulfonate groups are pH-independent. Marras et al. previously compared how the phase behavior of oligonucleotidecontaining complexes depends on the selection of PVBTMA versus poly(lysine) at various degrees of polymerization [14], illustrating the importance of molecular level details that are responsible for solid or liquid PEC formation.
In addition to the selection of starting materials, the condition of the external environment also plays a critical role in controlling the physical state of the resultant PECs. For instance, salt is commonly used as a stimulus to break and compensate the ion pairs between oppositely charged chains, and consequently, dissociate the PECs. For strongly interacting PECs that are initially solid precipitates, the addition of salt can transition solids into liquid coacervates and, ultimately, to homogenous solutions [20]. Following this established phenomenon, we employed sodium bromide (NaBr) salt to examine the phase transition of PVBTMA/PSS complexes through its complex/coacervate continuum. A full study of the rheological properties, phase behavior, and structure of this system is underway and remains outside the scope of this current work.
Although salt has been highly investigated as an additive over recent decades, the interplay between salt and solvent in PEC materials has received far less attention. In this study, we attempt to fill this gap by exploring how co-solvents (that introduce a hydrophobic constituent) and salinity together can tune the properties of the PVBTMA/PSS system. Selective binary mixtures of water and organic solvents enable a facile way to progressively modulate PEC behavior. Ethylene glycol and ethanol were deliberately selected as organic solvents due to their miscibility with water (Table 1). We first studied how solvent hydrophobicity alone influenced PEC properties by assembling the complex in salt-free ethylene glycol/water co-solvent conditions. Then we incorporated another dimension and added salt into the system to investigate the mutual effect of solvent and salinity. Optical microscopy was mainly used as the experimental approach to qualitatively map out the phase behaviors of these complexes. This study demonstrates how PECs, as a class of "smart" stimuli-responsive materials, can change their properties and respond to the external environment. We believe the results we present in this study will provide useful new insights into enriching and controlling the properties and functionalities of PECs through controlling the local solvent environment in complex materials.

Materials
The following chemicals were reagent grade and used as received unless otherwise specified: ethanol (HPLC Grade, Millipore Sigma), ethylene glycol (Reagent Plus, ≥ 99%, Millipore Sigma), (vinylbenzyl)trimethylammonium chloride (VBTMA, Sigma, 99%), poly(styrene sulfonate, sodium salt) (PSS, 201,700 g/mol, Polymer Standards Service), sodium b r o m i d e ( F i s h e r S c i e n t i f i c , > 9 9 % ) , 4 -c y a n o -4-(phenylcarbonothioylthio) pentanoic acid (CPhPA, Millipore Sigma), 2,2′-azobis[2-(2-imidazolin-2yl)propane]dihydrochloride (VA-044, Wako Chemicals, USA), acetic acid (glacial, Sigma, ≥ 99.85%), sodium acetate trihydrate (Sigma, ≥ 99%), and SnakeSkin dialysis tubing (MWCO 3.5 K, 22 mm, Thermo Scientific). All water used during the experiment was filtered from a Milli-Q water purification system at a resistivity of 18 Polymer synthesis The complex system studied here is formed by PVBTMA 100 and PSS 100 (Chemical structures are shown in Scheme 1 and the subscripts denote the degree of polymerization). PSS 100 was used as received (purchased from Polymer Standards Service). PVBTMA 100 was synthesized with aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization to be approximately symmetric to PSS, based on previous work in our group [29]. Desired amount of VA-044 initiator, VBTMA monomer, and CPhPA chain transfer agent (molar equivalence of 1 to 1000 to 10, respectively) were added to the acetate buffer solution in a dried 25-mL round bottom flask. The flask was then sealed, degassed with dried nitrogen, and heated at 50°C and constant stirring for at least 21 h. The reaction was then cooled to room temperature and opened to air and we obtained the crude pink polymer. The crude polymer was then dialyzed against Milli-Q water for 4 cycles of 8 h each. Lastly, the samples were lyophilized and we achieved ca. 2 g polymer in the end.

PEC sample preparation
PECs were prepared under 1:1 stoichiometric charge-matched conditions between polycation and polyanion. The "as prepared" polymer concentration was fixed at~1 wt% (10 mg/ mL). Following the protocol of the direct dissolution method [30], we added stock solutions of polycations and polyanions sequentially to a solution with desired amounts of NaBr stock solution (5 M) and co-solvent of Milli-Q water and ethylene glycol or ethanol. After all the solutions were added, samples were immediately vortexed for at least 30 s.

Optical light microscopy imaging
To visualize PECs directly at the microscale and determine the phases of these samples, we used optical phase contrast microscopy (Leica DMI 6000B with Leica Application Suite (LAS) image acquisition software, Wetzlar, Germany). PEC samples were first prepared in 1.5 mL microcentrifuge tubes, and immediately 100 μL of the well-mixed samples were transferred into ultra-low attachment 96-well plates (Costar, Corning Inc.). The plates were carefully sealed to prevent water evaporation. Imaging was performed 1 day after sample preparation to guarantee complete phase separation. Using ImageJ software, we later adjusted and enhanced the contrast of the acquired images for clarity.

Thermogravimetric analysis
After PEC samples were prepared in the 1.5 mL Eppendorf tubes, they were centrifuged at 4000×g for 15 min. Then 30 μL of the supernatant and around 5-mg complex materials were loaded onto aluminum pans whose weights were measured beforehand. After the samples were transferred, the weights of samples together with pans were recorded again. In this way, the mass of samples on the pans can be calculated. Next, the pans loaded with samples were carefully transferred into a Barnstead Thermolyne Furnace 1400. The furnace temperature was first set to 200°C and held for 2.5 h to evaporate all the solvents within the samples. The weights of pans with samples were measured again afterwards so that we were able to determine the weight of the evaporated solvents. After that, pans loaded with dried samples were put back into the furnace once again and heated at 600°C for 12 h. During this process, all the polymer contents were burned and removed. The weights were recorded for the last time, from which we can calculate the removed polymer mass and the mass of salt remains. For each condition, at least three different repeating samples were measured. Dixon's Q test was performed later to identify and discard outliers.

Salt-free PECs in ethylene glycol/water solvent mixtures
We first prepared PVBTMA/PSS complexes under salt-free conditions and characterized the resultant assembly morphologies with microscopy. Figure 1 shows a gallery of optical images of PECs assembled in increasing co-solvent ratios of ethylene glycol to water. As the ethylene glycol content increases, there was no evident change in the physical appearances of samples, which remained dense, amorphous, and opaque solid aggregates. This suggests that any potential co-solvent effect on the PEC material was not visually detectable on the micron scale. To support this observation, we then conducted thermogravimetric analysis (TGA) to quantify the exact distribution of the total solvent (water and ethylene glycol), polymer, and counterion components within the complex. This thermal technique provides a straightforward way to quantitatively measure the relative weights of the liquid, polymer, and salt in the PEC phase [27]. As shown in Fig. 2, the weight percentage of these three components were invariant to the addition of ethylene glycol in the binary solvent mixture, thus confirming the previous conclusions from the microscopy images that addition of co-solvent does not itself disrupt the complex.

NaBr doped PECs in ethylene glycol/water solvent mixtures
Next, we incorporated externally added salt into the PEC assemblies and observed the resultant behavior by microscopy. In the series of PECs in pure water only (i.e., without any ethylene glycol as a co-solvent), a phase transition of this system from solid to liquid was qualitatively mapped by a visual change in their physical appearances in between 2.0 and 2.5 M NaBr, from cloudy and dense aggregates into a fluid-like transparent network (Fig. 3). In other words, the advent of a liquid state can be judged through the emergence of clear structures on microscopy images. This method of determining physical states of this PEC has been confirmed by rheology measurements, which we will discuss in a forthcoming paper.
Although there were no noticeable differences in the PEC as co-solvent effects were introduced in the previous salt-free case, we notice an interesting trend as salt was brought into the PEC system containing ethylene glycol. In the gallery of optical images arranged in Fig. 3, if we navigate vertically from top to bottom, the fraction of ethylene glycol in the co-solvent gradually increases from 0 to 0.7, and accordingly, the solvent environment becomes more hydrophobic for the PEC phase. As marked by the solid red line, the solid-to-liquid phase transition occurred at lower salt conditions with increasing ethylene glycol content. Additionally, the critical point in salt resistance, where the two-phase PEC system turns into a onephase, homogenous polyelectrolyte solution across the binodal phase boundary, was also lowered as ethylene glycol content increased. This is marked by the solid blue line in Fig.  3. Overall, this set of experiments demonstrates how two orthogonal parameters can be used to transverse the complexcoacervate continuum state space.

NaBr doped PECs in ethanol/water co-solvent mixtures
To further expand on the intriguing findings shown in Fig. 3, we prepared otherwise identical PEC samples using ethanol as a co-solvent with water instead of ethylene glycol. As shown in Fig. 4, the same overall pattern was identified by microscopy. Both phase transitions can be carefully tuned as a function of ethanol fraction and NaBr salt concentration. Furthermore, when directly comparing these two solvent choices, we  We hypothesize that this difference between ethylene glycol and ethanol is due to the physical properties of these two solvents that influence the associative driving force and salt resistance of this PEC system in water. For instance, the relative polarities of ethanol and ethylene glycol are 0.654 and 0.79, respectively [28]. Therefore, under the same ratio of organic solvent and water, ethanol/water co-solvent creates a more "hydrophobic" environment for the complex phase,  causing the bulk complex to be less salt resistant. It is worth pointing out here that our observation is the opposite of what has been mentioned in previous publications. Chang et al. [31] have reported a dramatic increase in salt resistance for polypeptide-based coacervates when solvent was switched from water to a more hydrophobic mixture of isopropanol and water. Similarly, Sun et al. [32] have discovered that as the co-solvent became more hydrophobic, there was a slight increase in the salt concentrations needed to drive solid-to-liquid phase transition and complete dissociation of polysaccharide-based PECs. We think this striking difference between our work and previous studies came from the difference in the nature of polymer materials: PVBTMA and PSS are very hydrophobic, while both polypeptides polysaccharides are quite hydrophilic. Nevertheless, there are clearly other subtle factors that govern the effects of solvent on PECs.
More rigorous investigations to test the universality of what we have observed in these two limiting cases are underway.

Conclusion
In summary, by studying comprehensively the assembly of a model PEC system under various co-solvent and salinity conditions, we report a straightforward method of modulating the complex stability and responsivity. To this end, PVBTMA and PSS were ideal since this strong polycation-polyanion pairing resulted in solid precipitates that exhibited high salt resistance in water for systematic co-solvent and salt studies.
When we introduced co-solvents of ethylene glycol and ethanol, both solid-to-liquid and liquid-to-solution phase transitions were tunable and occurred at much lower salt concentrations. This finding suggests that co-solvents can be exploited as a powerful agent to modulate the behavior of strongly interacting PECs upon the addition of salt. Furthermore, by progressively adjusting the co-solvent ratios, we were able to demonstrate a clear correlation between salt resistance and solvent quality: aqueous solvent mixtures can weaken the salt resistance of PEC systems in a systematic manner.
Altogether, this work provides practical insights into enriching our understanding of the phase behavior along the coacervate/complex continuum. Future work in our group will expand on the fundamental mechanisms and molecular details behind this physical behavior for charge-driven assemblies. For amphiphilic surfactants and polymers, solvent quality is a known important factor for both the thermodynamics and kinetics of these self-assembled systems. The extension of this general idea for charged polymers has not yet been fully realized, but careful examination of controlled systems can elucidate important structure-property relationships for the continued integration of PECs into advanced materials applications. Matthew V. Tirrell 's research has been in the fields of polymer interfaces, dynamics, fluid phase behavior, and nanomedicine. He is particularly known for his work on polymer brushes, surface force measurement, peptide amphiphiles, and polyelectrolyte complex phase behavior. Ph.D. students and 50 postdoctoral researchers. Professor Tirrell is a member of the National Academy of Engineering, the National Academy of Sciences, the American Academy of Arts and Sciences, and the Indian National Academy of Engineering, and is a Fellow of the American Institute of Medical and Biological Engineers, the AAAS, and the American Physical Society. It is a pleasure to make this contribution to a journal issue recognizing the many contributions of Matthias Ballauff to the science of macromolecules. Professor Ballauff has been a friend, scientific collaborator, and valued colleague for more than 30 years. All of his colleagues have enjoyed and learned from our associations with him.