Polyelectrolyte solubility in different buffer systemsInitially, εPolyK, αPolyK, Protamine, PolyD, and HA with similar ionization degrees and molecular weights (3–7 kDa) were prepared, as outlined in Fig. 1. More specifically, to prepare synthetic polymers with the same number of charged moieties, PMAA and PAEMA with a degree of polymerization (DP) of 40 were employed. A well-controlled DP was successfully obtained for each polymer with a desired monomer conversion, DP, and polydispersity index (PDI) being confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC) (Supplementary Figs. 1–2). To assess the impact of the polyelectrolyte hydration properties on complex coacervation, the solubility of each polyelectrolyte was measured in an aqueous buffer (Table 1). Since the polymer solubility can be influenced by a wide range of factors, such as the temperature, pH, salt content, and buffer composition, a 19 mM Tris-chloride (Tris-Cl) buffer at pH 7.5 was employed due to its ability to dissolve all polyelectrolytes at room temperature. Saturated solutions were prepared by collecting the supernatants of the supersaturated polyelectrolyte solutions after centrifugation. The solubility of each polyelectrolyte was quantified by measuring the weights of the dried saturated solutions of specific volumes. It was found that the solubilities of εPolyK (326.67 mg/mL) and αPolyK (>1000 mg/mL) were approximately one order of magnitude higher than that of PAEMA (63.33 mg/mL), despite both sharing the same ionic functional group. This suggests that εPolyK and αPolyK, which each possess a peptide backbone, are more hydrophilic than PAEMA, which possesses an aliphatic hydrocarbon backbone. Additionally, the higher solubility limit of αPolyK compared to that of εPolyK clearly demonstrates the influence of the backbone hydrophilicity, since these two structures exist as two different isoforms of the same species. In the case of protamine, at concentrations >40 mg/mL, simple coacervation occurred, characterized by a spherical shape, indicating that phase separation driven by hydrophobic arginine–arginine stacking had taken place21. The occurrence of simple coacervation in protamine, rather than precipitation, suggests that protamine exhibits a greater degree of hydration24. Nonetheless, it should be noted that coacervation is more favored in its salt form (i.e., protamine sulfate), as shown in Supplementary Fig. 3. In the case of dialyzed protamine (protamine sulfate thoroughly dialyzed against deionized water), its coacervation assembly in the 19 mM Tris-HCl buffer pH 7.4 was also observed using a microscope. At 40 mg/mL, the dialyzed protamine still underwent coacervation, although the yield of coacervates was smaller than that observed in the case of protamine sulfate. We also confirmed that coacervation is more dominant at a higher concentration of 100 mg/mL. This demonstrates that although protamine on its own undergoes simple coacervation above 40 mg/mL, the impact of counter ions (i.e., sulfate) should also be considered. Among the anionic polyelectrolytes, PMAA, PolyD, and HA exhibited significantly higher solubilities than the cationic polyelectrolytes, exceeding 1000 mg/mL. However, measuring the solubility beyond 1000 mg/mL was not feasible because of the large volume of polymer powder required, and the high viscosity of the resulting solution.Fig. 1: Chemical structures of the selected polyelectrolytes.The asterisk (*) indicates the primary sequence of protamine. The values n1 = 40, n2 = 25–35, n3 = 30, n4 = 40, n5 = 30, and n6 = 1.2–25 correspond to the respective polymer repeating units.Table 1 Average degrees of polymerization, molecular weights, solubilities, and SASA values of the polyelectrolytesSubsequently, density functional theory (DFT) calculations were employed to further explore the solubility differences between polyelectrolytes. This involved calculating the optimized structures and atomic charges of the polyelectrolytes. Their solvent accessible surface areas (SASAs) were also determined, along with their atomic charge distributions. Additional details are provided in the “Methods” section.Based on the calculation results, the main difference between the cationic polyelectrolytes was determined to be the optimized structure. More specifically, PAEMA exhibits a short backbone with long, radiating side chains, while εPolyK features a long backbone and no side chains, resulting in a linear elongated shape (Supplementary Fig. 4a and b). This leads to different levels of accessibility in a solvated environment, wherein εPolyK exposes nearly all atoms, while PAEMA, which contains many overlapping areas, exposes relatively fewer atoms, as verified by the computed SASA values (Table 1 and Supplementary Fig. 5). More specifically, the larger SASA value of εPolyK (1309 Å2) compared to that of PAEMA (953 Å2) indicates that εPolyK is more hydrated. Thus, despite their identical charged groups, the different backbone shapes render εPolyK more soluble than PAEMA. Similar to PAEMA, poly-arginine, a simplified model that was used herein to represent protamine, possesses a short backbone bearing long side chains (Supplementary Fig. 4c). However, due to its β-sheet-like structure, the overlapping areas are expected to be much smaller, as evidenced by its SASA value, which is comparable to that of εPolyK (Table 1). Poly-arginine is therefore expected to maintain its hydrated state without undergoing precipitation. Moreover, the amphiphilic and quasi-aromatic properties of the arginine guanidine group likely promote π stacking and lead to the formation and stabilization of simple coacervation21,25,26. In the cases of εPolyK (1309 Å2) and αPolyK (1219 Å2), only a minor difference was observed between their SASA values. This was likely due to the limitations of the pentamer system since discernable differences were observed in the experimental results (as detailed in the subsequent sections).For the anionic PMAA and PolyD species, no significant variations were found for their optimized structures or SASA values (Table 1 and Supplementary Fig. 6a and b). However, their atomic charges differed, demonstrating that the peptide backbone of PolyD possesses a distinct and wider range of charges than the hydrocarbon backbone of PMAA (Supplementary Fig. 7). Such a discrepancy in the charge distribution, which originated from the backbone type, will therefore be expected to influence the interaction strengths between these species and the cationic polyelectrolytes, ultimately leading to different coacervation behaviors. It was also found that the optimized structure of hyaluronic acid (HA) was linear and that the SASA value of this species was exceptionally large (Table 1 and Supplementary Fig. 6c). Thus, the large accessible area of HA, along with its abundant hydroxyl groups, should promote interactions with water molecules and ions to render HA highly soluble.Effect of the polycation:polyanion ratio on the phase behaviorSubsequently, the coacervation phase behavior was observed as a function of the cationic:anionic polyelectrolyte mixing ratio using 10 mM Tris-Cl buffer (pH 7.5, Fig. 2a–e). This assessment was conducted by measuring the relative turbidity and by observing the liquid–liquid phase separation using an optical microscope.Fig. 2: Complex coacervation behaviors in different buffers.Schematic representation (top) and plots of turbidity vs. the polymer mixing ratio (bottom) for phase separation. a–e In Tris-Chloride (Tris-Cl) buffer; (f–j) In Tris-Acetate (Tris-Ac) buffer; (k–o) In Tris-Phosphate (Tris-Po) buffer. Black plots = PAEMA:PMAA pairs; purple plots = εPolyK:PMAA pairs; orange plots = Protamine:PMAA pairs; blue plots = PAEMA:PolyD pairs; green plots = PAEMA:HA pairs. In the phase diagrams, closed markers indicate the mixing ratios wherein phase separation was observed using an optical microscope. The open markers indicate the mixing ratios wherein phase separation did not occur. The X marks indicate the mixing ratios wherein aggregation was observed. The gray region in each phase diagram represents the coacervation region where phase separation of the polyelectrolytes occurs. Each data point is based on at least three replicate experiments carried out for each respective polyelectrolyte.In the PAEMA:PMAA, εPolyK:PMAA, and Protamine:PMAA systems, precipitates were observed by optical microscopy at all mixing ratios evaluated herein (marked with an “X” in Fig. 2a–c, see also Supplementary Figs. 8–10). However, for the PAEMA:PolyD and PAEMA:HA systems, broader coacervate ranges were observed (marked as filled circles in Fig. 2d and e, see also Supplementary Figs. 11 and 12).In the cases of the PAEMA:PMAA, εPolyK:PMAA, and Protamine:PMAA systems in 10 mM Tris-Cl buffer, precipitation occurred at all mixing ratios, rendering it difficult to observe any coacervation tendencies. To address this, a higher-ionic-strength buffer was employed (19 mM Tris-Cl buffer, pH 7.5) to investigate the formation of coacervates through charge screening (Supplementary Fig. 13). Under these conditions, coacervates were formed at narrow regions where, in most cases, the turbidity reached a maximum value (marked as filled circles in Supplementary Fig. 13a–c). This aligns with a previous study showing that coacervates are dominant at their maximum and net charge points27,28. For the majority of other mixing ratios, precipitates were noticeable under optical microscopy observations (marked with an “X” in Supplementary Fig. 13a–c). Interestingly, the αPolyK:PMAA pair formed coacervates at all phase-separated mixing ratios, highlighting the substantial influence of the backbone hydrophobicity on the phase behaviors of such polyelectrolyte complexes (Supplementary Fig. 14e and h). In the case of the PAEMA:PolyD and PAEMA:HA pairs, coacervates were observed at almost all phase-separated mixing ratios; however, their phase separation propensities decreased compared to those observed in the 10 mM buffer, and this was attributed to the increased ionic strength.Given that the tight binding of molecules and the corresponding expulsion of water and counterions leads to precipitation, the coacervate phase region can provide insights into the hydrophobicity of a polyelectrolyte. While a direct comparison of the solubilities of PMAA, PolyD, and HA poses challenges due to their high solubilities (i.e., >1000 mg/mL), the observed coacervate phase regions suggest that both PolyD (with an amide backbone) and HA (with a carbohydrate backbone) are more hydrophilic than PMAA (with an aliphatic chain). Notably, this observation correlates with the DFT calculation results. Moreover, when comparing the coacervate phase regions of εPolyK:PMAA and εPolyK:HA, in addition to those of Protamine:PMAA and Protamine:HA, in 19 mM Tris-HCl buffer pH 7.4 (Fig. 2b and c, see also Supplementary Fig. 15), it was clear that coacervate formation was more favored in the HA combinations. This preference is likely due to the higher hydrophilicity of HA (Supplementary Fig. 15). This can be further supported by the refractive index and average droplet size (Supplementary Fig. 16), which showed a tendency toward a negative correlation. This implies that coacervates with larger sizes, such as the PAEMA:HA and PAEMA:PolyD pairs, are more hydrated, resulting in a lower refractive index29. These results therefore demonstrate that the mixing ratio of oppositely charged polyelectrolytes affects the phase behavior, and that the solubility of the polyelectrolyte plays a crucial role in determining the coacervate region.Effect of the buffer anions on complex coacervationTo further investigate the impact of buffer ions on complex coacervation, different salts were introduced into the Tris buffer, namely chloride, acetate, and phosphate, denoted as Tris-Cl, Tris-Ac, and Tris-Po, respectively (10 mM, pH 7.5). By analyzing the turbidity and microscopic images, shifts in the coacervate and precipitate phase regions of certain polymer pairs were observed (Fig. 2 and Supplementary Figs. 8–12). In all cases, the liquid coacervate regions expanded in the order: Tris-Cl < Tris-Ac < Tris-Po buffers. More specifically, in the case of the PAEMA:PMAA, εPolyK:PMAA, and Protamine:PMAA pairs, the precipitate phase dominated when dissolved in the Tris-Cl buffer. However, when dissolved in the Tris-Ac buffer, the coacervate regions were broadened. Intriguingly, in the Tris-Po buffer, all precipitate regions vanished, and coacervates formed at all phase-separated ratios. Moreover, the PAEMA:PolyD and PAEMA:HA pairs predominantly formed coacervates in the Tris-Cl buffer, although precipitates were observed at certain mixing ratios. Similar to the behavior of the PAEMA:PMAA, εPolyK:PMAA, and Protamine:PMAA pairs, in both the Tris-Ac and Tris-Po buffers, the PAEMA:PolyD and PAEMA:HA pairs exclusively formed a coacervate phase (Fig. 2d, i, n, e, j, o, and Supplementary Figs. 11 and 12). Considering that a broader coacervate phase region is indicative of hydrated and loosely formed complexes, these results are consistent with the solubility outcomes. Specifically, the significantly broader coacervate region observed for the εPolyK:PMAA pair compared to the PAEMA:PMAA and Protamine:PMAA pairs correlates with the higher solubility of εPolyK. Notably, the observed increase in the coacervate phase region according to the order chloride < acetate < phosphate, represents an inversion of the Hofmeister series30.Effect of the salt on complex coacervationAs mentioned previously, electrostatic interactions play a critical role in driving complex coacervation. Owing to the screening effect of salts on the polyelectrolyte charges, the addition of a salt induces disassembly of the complex coacervation system. The amount of NaCl required to dissolve the coacervates (referred to as the salt resistance, C*) depends on the interaction strength between the polymers. Thus, the salt resistance of each polymer coacervate pair formed at the optimal stoichiometry was determined by adjusting the NaCl concentration until the turbid coacervate suspension (indicated by filled marks) transformed into a uniform liquid phase (indicated by open marks) (Fig. 3). From this point onward, the experiments were conducted using 19 mM Tris-Cl buffer at a mixing ratio favoring coacervate formation rather than precipitation. The complex coacervation of the PAEMA:PMAA pair exhibited the highest salt resistance, remaining stable up to a NaCl concentration of 1.5 M. The salt resistance of the other pairs followed the order: εPolyK:PMAA at 1.2 M NaCl > Protamine:PMAA at 1.0 M NaCl > αPolyK:PMAA and PAEMA:PolyD at 0.8 M NaCl > PAEMA:HA at 0.5 M NaCl.Fig. 3: Salt resistance of the complex coacervates.a Turbidity results obtained across various sodium chloride salt concentrations. The areas filled with colors indicate the corresponding coacervate regions. b NaCl salt resistance characteristics for the various coacervate pairs. In each case, the polymer concentration (Cp) was 1 mg/mL, and the samples were analyzed immediately after complexation. The error bars indicate the standard deviations determined from three separate measurements.Overall, the obtained results demonstrated a negative correlation between the solubility and simulation outcomes. The maintenance of coacervates at higher salt concentrations indicates that complexation and coacervation contributed not only to the electrostatic attractions but also to other non-ionic interactions. To investigate the impact of hydrophobic interactions, 10% 1,6-hexanediol was introduced into each coacervate pair dissolved in 19 mM Tris-Cl buffer (pH 7.4) containing 100 mM NaCl (Supplementary Fig. 17). The reduction in ionic strength caused by the presence of NaCl was expected to facilitate the observation of hydrophobic-driven coacervates. In addition, the presence of 1,6-hexanediol, a weak hydrophobic interaction disruptor31,32, was anticipated to provide insights into the role of hydrophobic interactions in coacervate formation. In the case of the PAEMA:PMAA pair, coacervate deformation was observed after the addition of 1,6-hexanediol. This indicates that hydrophobic interactions stemming from their hydrophobic aliphatic backbones played a significant role in driving coacervate formation. Conversely, in the case of the highly hydrated PAEMA:HA pair, the contribution of the hydrophobic interactions to maintaining LLPS seemed to be weaker. Taking the above results into account, the PAEMA:HA pair was considered to be mainly dependent on electrostatic interactions. Additionally, it was observed that αPolyK:PMAA coacervates bearing a shorter backbone chain disappeared at a lower NaCl concentration than the ɛPolyK:PMAA coacervates (Supplementary Fig. 14), indicating that a more hydrophobic backbone contributes to an improved salt resistance. This agrees with previous findings, in which the phase separation of complex coacervates is also dependent on the hydrophobic interactions present in a high-salt regime33.Coalescence behaviors of the complex coacervatesHaving demonstrated that both the polymer backbone and the ionic functional groups can influence the phase separation properties of complex coacervates, the ability of these polyelectrolyte characteristics to be manifested in the viscoelastic properties of the resulting coacervates was evaluated. Thus, to characterize the viscoelastic properties of the coacervates, the coalescence behaviors of five pairs of complex coacervates were observed, focusing on the mixing ratios at which the coacervates exhibited the maximum turbidity. As shown in Fig. 4a, when the two liquid droplets come into contact, they merge into a single ellipsoidal liquid body, which subsequently relaxes from this deformed state to yield a spherical shape. The linear slopes obtained for the relaxation time curves of the coalescing coacervates were then used to determine the ratio (η/γ) of the viscosity (η) to the surface tension (γ), which is otherwise known as the inverse capillary velocity (Fig. 4b).Fig. 4: Coalescence behaviors of the complex coacervates.a Representative images of the complex coacervate droplets formed in the 19 mM Tris-Cl buffer. For imaging purposes, all liquid/condensate pairs were prepared at the maximum fixed total polymer concentration (i.e., Cp = 1 mg/mL). The time scale units are seconds (s), and the scale bars are 5 μM in all images. b Relaxation time vs. length scale for each polymer pair. The inverse capillary velocity values (s/μm) are indicated for each pair. All samples were prepared using the optimal stoichiometry. Each data image is representative of the observed behavior from at least three test replicates of each respective polyelectrolyte pair.It was found that the coacervates formed by the aliphatic synthetic polymer pair (i.e., PAEMA:PMAA, ~38.85 s/μm) exhibited an approximately three-fold slower fusion than the εPolyK:PMAA coacervates (~13.92 s/μm), wherein εPolyK shares the same ionic functional groups as PAEMA but possesses a peptide backbone. Additionally, the coacervates of the Protamine:PMAA pair exhibited an approximately two-fold slower fusion (~29.90 s/μm) than that of the εPolyK:PMAA coacervates. This finding agrees with previous literature21, suggesting that arginine is more hydrophobic than lysine and that it contributes to the enhanced physical properties of arginine-rich coacervates. Similarly, despite the fact that PolyD shares the same ionic functional groups as PMAA and possesses a peptide backbone, the PAEMA:PolyD coacervates (~0.06 s/μm) exhibited a significantly faster rate of fusion than the PAEMA:PMAA coacervates (~38.85 s/μm). This result indicates that more hydrated peptides undergo faster fusion than aliphatic synthetic polymers. Furthermore, it was found that the coacervates of the PAEMA:HA pair fused quickly with an inverse capillary velocity of ~0.12 s/μm. Since the hindered relaxation process represents the solid-like properties of a coacervate with viscoelastic properties, this finding highlights the influence of the hydration properties of the polyelectrolyte backbone on the material properties of the resulting coacervate.
