Effect of mixed solvents on polyelectrolyte complexes with salt

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 bromide (Fisher Scientific, > 99%), 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPhPA, Millipore Sigma), 2,2′-azobis[2-(2-imidazolin-2-yl)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 18.2 MΩ cm at 25 °C. The acetate buffer solution was prepared with 0.1 M acetic acid and 0.1 M sodium acetate trihydrate (0.1 M) (42/158, v/v) at pH 5.2.

The complex system studied here is formed by PVBTMA₁₀₀ and PSS₁₀₀ (Chemical structures are shown in Scheme 1 and the subscripts denote the degree of polymerization). PSS₁₀₀ was used as received (purchased from Polymer Standards Service). PVBTMA₁₀₀ 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.

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.

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.

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.

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 one-phase, 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 complex-coacervate continuum state space.

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 noticed that at equivalent ratios of organic solvent to water, both phase transitions with ethylene glycol occurred at higher NaBr salt concentration levels than with ethanol. For example, at 4:6 organic solvent to water mixtures, for ethylene glycol, solid precipitates converted into liquid coacervates at 0.5–1.0 M NaBr and into solution at 1.5–2.0 M NaBr. For ethanol, these transition points were measured to be 0–0.5 M NaBr and 1.0–1.5 M NaBr, respectively.

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.