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. 2026 Feb 1;6(2):881–892. doi: 10.1021/jacsau.5c01238

Sticker-Spacer Molecular Design Controls Coacervate Formation and Internal Microenvironments in Low-Molecular-Weight Compounds

Sayuri L Higashi †,‡,§,*, Koichiro M Hirosawa ∥,⊥, Ryutaro Fujimoto #, Kodai Kanemaru ∇,○, Norio Yoshida ∇, Kenichi G N Suzuki ∥,⊥, Masato Ikeda ‡,§,⊥,#,◆,*
PMCID: PMC12933334  PMID: 41755842

Abstract

Coacervates formed via liquid–liquid phase separation have been established as potential protocells in life studies. Unlike widely studied macromolecules, coacervates from low-molecular-weight (M w) compounds have recently gained importance because they provide simple but valuable in vitro models for biomolecular condensates and serve as promising platforms for the development of functional biomaterials. Herein, we present a modular molecular design for the phase separation of low-M w compounds containing two aromatic or cycloalkane stickers linked via a flexible hydrophilic spacer. These low-M w compounds self-assemble into micrometer-scale liquid-like coacervates at submillimolar concentrations. The coacervates provide a hydrophobic internal microenvironment that can selectively sequester hydrophobic guest molecules while excluding hydrophilic molecules. We demonstrate the controlled release of hydrophobic drugs encapsulated in reduction-responsive coacervates composed of nitrophenyl groups as stickers that are cleaved by the addition of a reductant to induce the disassembly of the coacervates. Importantly, subtle variations in the chemical structures of the sticker groups resulted in significant differences in the internal microenvironments of the coacervates. Delicately balanced coacervates facilitate the interfacial accumulation of polysaccharides bearing appropriate fluorescent dyes, which effectively stabilize the coacervates against size expansion due to coalescence. This research highlighting a rational molecular design for the construction of modulable simple coacervates composed of low-M w compounds offers an opportunity to explore bioapplications as biofunctional soft materials and construct more complicated coacervate-based protocell models.

Keywords: coacervates, liquid−liquid phase separation, self-assembly, stimuli responsiveness, supramolecular soft materials

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Introduction

Compartmentalization plays a crucial role in the spatiotemporal regulation of numerous biological functions in living systems. Many synthetic compartments have been designed using bottom-up approaches to create cell-like materials. − Coacervates are formed spontaneously through the liquid–liquid phase separation (LLPS) of typical polyelectrolytes, such as nucleic acids and polypeptides, via multiple noncovalent interactions. , LLPS-induced coacervates have attracted considerable attention as mimics of protocell models relevant to the origin of life − and membraneless organelles, , which are also known as biomolecular condensates. , Although many studies have involved synthetic coacervates composed of various polyelectrolytes, their biocompatibility and applicability in living systems are limited by a lack of stability under biological conditions due to their high charge density and complex molecular structures. Recently, several research groups have synthesized phase-separating molecules based on low-molecular-weight (M w) compounds to create synthetic coacervates with multiple functions, including selective guest-molecule partitioning and acceleration of chemical (enzymatic) reactions. The simplification of molecular design could address the aforementioned challenges in coacervates and enable the implementation of new functions, such as responsiveness to specific stimuli, e.g., pH, − light, , and redox reactions, ,− by incorporating stimuli-responsive molecular components, which is a well-developed strategy in the field of supramolecular hydrogels. , Recent studies have revealed that the sticker-spacer motif is an important structure in phase-separating molecules. ,,,,,− Simple coacervates composed of fluorescent pyrene sticker-spacer-type molecules have been developed and applied for protein delivery into living cells. The authors synthesized and evaluated five additional sticker-spacer compounds; however, only one, which used dimeric naphthyl spacers, formed similar coacervates. Investigating coacervate-forming molecules will aid in the development of new synthetic coacervates tailored for various applications. However, a deeper understanding of the physical properties, particularly the internal chemical environments, of guest partitioning depending on the molecular structures of the coacervate components remains scarce.

In this study, we designed and synthesized several sticker-spacer-type molecules with varying hydrophobic sticker parts (phenyl groups or linear or cyclic aliphatic groups) and investigated their impact on the formation of coacervates (Figure a) and their physical properties, including water content, fluidity, chemical stability, selective partitioning of guest molecules, and stimuli-responsive functions. Although all coacervates exhibited hydrophobic internal environments, detailed characterization revealed that sticker variation resulted in significant differences in the internal dynamics. Remarkably, polysaccharides bearing hydrophobic tetramethylrhodamine (TMR) anchors showed dramatic differences in localization–interior or the surface of coacervates–depending on sticker variation between phenyl groups or cyclic aliphatic group. These internal heterogeneities were further elucidated using total internal reflection fluorescence microscopy (TIRFM) and all-atom molecular dynamics (AAMD) simulations. Our findings provide important insights for the design of functional soft materials consisting of hierarchical structures, including coacervates, as key components of living cells.

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(a) Chemical structures of OEG-bis-X (X = NPmoc, Pmoc, cHex) and schematic of coacervation. (b) Turbidity (OD600) of solutions containing OEG-bis-X at different concentrations in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1). Error bars indicate the standard deviation (SD) of the average OD600 (N = 3). (c) Representative confocal laser scanning fluorescence microscopy (CLSM) images (top: DIC, bottom: fluorescence, before and after fusion) of the OEG-bis-X coacervates. [OEG-bis-X] = 4.3 mM and [Nile red] = 5.0 μM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. Scale bar: 10 μm. (d) Representative CLSM image of PEG-passivated green fluorescent particles encapsulated within an OEG-bis-X coacervate. Scale bar: 5 μm. (e) MSD vs lag time. MSD data for individual particles from a single droplet are plotted. The black solid line has a slope of 1. Data were collected from three independent coacervates (N = 3) for each OEG-bis-X, and individual plots show the averaged trajectories of multiple particles within each coacervate. (f) FRAP analysis showing the recovery of the fluorescein-allyl fluorescence within the coacervates after photobleaching. [OEG-bis-X] = 4.2 mM and [fluorescein-allyl] = 5.0 μM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. Scale bar: 5 μm. Data are presented as the mean ± the SD (N = 3).

Results and Discussion

Design and Synthesis of Phase-Separating Molecules and Their Coacervate Formations

To develop phase-separating molecules, we designed and synthesized sticker-spacer-type low-M w compounds, namely, OEG-bis-X (X = NPmoc: 4-nitrophenylmethoxycarbonyl, Pmoc: phenylmethoxycarbonyl, and cHex: cyclohexylmethoxycarbonyl, Scheme S1). Flexible and hydrophilic oligoethylene glycol (OEG) was used as the spacer, with X as the potential sticker at both ends connected by carbamate bonds (Figure a). The compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (43 mM). When each OEG-bis-X DMSO stock solution was vigorously mixed with 100 mM phosphate buffer (pH 7.5) at ambient temperature, the solution immediately turned turbid (Figure b), and spherical structures with diameters of up to several micrometers were clearly observed under an optical microscope (Figure c). The micrometer-sized spherical structures, which can be defined as coacervates (vide infra), formed above a certain threshold concentration (∼0.5 mM) (Figure S17). The concentration of phosphate ions employed as buffering components affected the size of the coacervates; i.e., smaller coacervates were formed at higher phosphate concentrations (Figure S18). In addition, we evaluated the effect of DMSO on coacervation by varying the DMSO content when mixing the stock DMSO solution of OEG-bis-X with aqueous buffer (0, 1, or 10 vol %). For all three OEG-bis-X cases, the coacervate populations at 1 vol % DMSO were lower than those at 10 vol % with smaller coacervates and lower turbidity. Furthermore, mixing OEG-bis-NPmoc with an aqueous buffer containing no DMSO did not produce a cloudy dispersion even after ultrasonication. However, a heat-and-cool treatment of this mixture gave a cloudy dispersion containing coacervates observed by microscopy (Figure S19). In contrast, mixing OEG-bis-Pmoc or OEG-bis-cHex with aqueous buffer containing no DMSO yielded cloudy dispersions containing micrometer-sized coacervates after ultrasonication. We used the 10 vol % DMSO condition as the standard protocol in this study. Importantly, the stochastically contacted coacervates spontaneously fused into larger coacervates within several tens of milliseconds (ms), as shown in Figure c (bottom), indicating liquid-like properties, and they exhibited Brownian motion, as expected for their sizes. In addition, wetting of the cover glass surface was frequently observed for the OEG-bis-X coacervates during microscopic observations (Figure S20).

To investigate the driving forces of coacervation, we additionally surveyed the coacervate formation ability of OEG-bis-X bearing the stickers: nHex (n-hexyloxycarbonyl), 2EtHex (2-ethyl-hexyloxycarbonyl), nBut (n-butyloxycarbonyl), Alloc (allyloxycarbonyl), and moc (methoxycarbonyl) (Figure S21a). Because both OEG-bis-Pmoc (phenyl) and OEG-bis-cHex (cycloalkane) formed coacervates, π–π interactions may not be indispensable for coacervate formation, but their properties were different. OEG-bis-nHex, OEG-bis-nBut (linear alkanes), and OEG-bis-2EtHex (branched alkane) formed significantly smaller coacervates with an averaged diameter of 0.8 ± 0.5 μm (namely, mini-coacervates) compared to OEG-bis-cHex coacervates with an averaged diameter of 1.9 ± 0.8 μm (Figure S21b). This indicates that the cycloalkane structure is more effective for the formation of coacervates. Nevertheless, OEG-bis-nBut formed similar coacervates at higher concentrations (43 mM), whose microscopic morphologies were comparable to that of OEG-bis-cHex coacervates (Figures S21c and S22). In stark contrast, OEG-bis-moc did not form coacervates but gave only amorphous structures, which were rarely found at 4.3 and even 43 mM (Figure S22). Similarly, OEG-bis-Alloc showed no coacervate formation, even at 43 mM.

To gain insight into the effect of different sticker units (NPmoc, Pmoc, cHex, or moc) on phase separation, we performed AAMD simulations. Aggregation, which should be related to phase separation, occurred within 5 ns of simulation time for OEG-bis-X molecules bearing NPmoc, Pmoc, or cHex stickers (Figure S23). Here, liquid-like “aggregation” is used to describe the formation of molecular assemblies driven by hydrogen bonding between OEG-bis-X molecules (i.e., solute–solute) and those with water molecules [i.e., solute–solvent (water)], as well as hydrophobic interactions between the sticker moieties (X) of OEG-bis-X, which differed from solid-like aggregates without water molecules. In contrast, OEG-bis-moc did not exhibit aggregation under the same simulation conditions. These findings are consistent with the effect of sticker units on the formation ability of coacervates.

Among the OEG-bis-X variants tested, only OEG-bis-NPmoc, OEG-bis-Pmoc, and OEG-bis-cHex formed micron-sized coacervates (Figure ). To gain deeper insight into their physicochemical properties, we investigated these three coacervates in detail. To measure the viscosity inside the OEG-bis-X coacervates, we conducted a microrheological analysis using polyethylene glycol (PEG)-passivated fluorescent particles with a radius of 0.5 μm. The fluorescent particles were encapsulated within the OEG-bis-X coacervates (Figure d), and their trajectories were analyzed over time to obtain mean-squared displacement (MSD). The MSD plots revealed a diffusion exponent of α ≈ 1, consistent with normal diffusion due to Brownian motion in a purely viscous medium (Figure e). The diffusion coefficients D varied depending on the type of stickerNPmoc, Pmoc, or cHexand were determined to be 5.28 × 10–7 (95% confidence interval (Cl): 4.05–6.78 × 10–7), 4.54 × 10–6 (95% Cl: 3.91–5.25 × 10–6), or 1.55 × 10–6 (95% Cl: 1.36–1.77 × 10–6) m2 s–1, respectively. Using the Stokes–Einstein equation (D = k B T/6πηa, where k B T is the thermal energy scale, η is the viscosity, a is the radius of particles), we calculated the corresponding viscosities: η NPmoc = 0.83 Pa·s (95% Cl: 0.62–1.04 Pa·s), η Pmoc = 0.097 Pa·s (95% Cl: 0.083–0.112 Pa·s), and η cHex = 0.28 Pa·s (95% Cl: 0.25–0.31 Pa·s). These values are comparable to those of glycerol or light syrup, but considerably lower than that of typical protein-based coacervates. ,

We also examined the molecular dynamics of the coacervates using fluorescence recovery after photobleaching (FRAP) experiments with the coacervates stained with fluorescein-allyl. After photobleaching a portion within the coacervates of OEG-bis-NPmoc, OEG-bis-Pmoc, and OEG-bis-cHex, rapid fluorescence recovery was observed, and the 50% fluorescence recovery times were estimated to be 2.2, 0.6, and 1.2 s, respectively (Figure f). In addition, the water contents of the OEG-bis-NPmoc, OEG-bis-Pmoc, and OEG-bis-cHex coacervates were 39 ± 4.1%, 68 ± 9.2%, and 52 ± 17%, respectively (Table S1), which is within the typical range reported for coacervates (from 40 to 90%). These results indicate that OEG-bis-X can self-assemble into simple liquid-like coacervates under aqueous conditions, and that the internal environment of coacervates is strongly influenced by the sticker unit, resulting in higher water content and lower viscosity in the order of OEG-bis-Pmoc > OEG-bis-cHex > OEG-bis-NPmoc. Next, we investigated the stability of the OEG-bis-X coacervates against NaCl, 1,6-hexanediol, and urea for comparison with simple biomolecular condensates composed of proteins. − The addition of 5 M NaCl (final concentration of 1 M) barely affected the morphology of the three OEG-bis-X coacervates (Figure S24). In contrast, 40% (w/v) 1,6-hexanediol (20% (w/v) final concentration) induced the significant dissolution of the OEG-bis-Pmoc and OEG-bis-cHex coacervates under optical microscopic observations, which is consistent with liquid-like biomolecular condensates, while the OEG-bis-NPmoc coacervates were not significantly affected. Nevertheless, turbidity measurements revealed a decrease in the opacity of aqueous solutions of all three coacervates, indicating that 1,6-hexanediol induces structural destabilization in each case. Furthermore, both microscopic observation and turbidity measurements showed a decrease in coacervate size and turbidity, respectively, upon the addition of 15 M aqueous urea (final concentration of 5 M) in all three systems (Figure S24b–d). This suggests that the stability of these coacervates against denaturants such as urea is relatively low, indicating that intermolecular hydrogen bonding contributes to coacervate formation, similar to simple biomolecular condensates.

To further explore the driving forces such as hydrophobic and hydrogen bonding interactions underlying coacervate formation, we analyzed the results of the AAMD simulations of the OEG-bis-X molecules bearing NPmoc, Pmoc, cHex or moc. We first analyzed the time evolution of hydrogen bonding interactions among the OEG-bis-X molecules, water, and DMSO within the aggregates in the AAMD trajectories (Figure S25). Compared with OEG-bis-moc, which did not exhibit aggregation, OEG-bis-NPmoc, OEG-bis-Pmoc, and OEG-bis-cHex showed fewer hydrogen bonds with water and more intermolecular hydrogen bonds between OEG-bis-X molecules, which are considered to be crucial for aggregation. The number of hydrogen bonds between OEG-bis-X and DMSO (denaturant) was consistently low across all cases, suggesting that DMSO is largely excluded from the coacervate interior after phase separation. We also analyzed the time-resolved distribution of the center-of-mass distances between stickers (Figure S26). For OEG-bis-cHex, a significant fraction of sticker pairs remained at ∼6 Å throughout the simulation. In contrast, OEG-bis-NPmoc and OEG-bis-Pmoc showed frequent interactions at 4–5 Å, characteristic of π–π stacking. Particularly, OEG-bis-NPmoc exhibited a notable population at ∼4 Å, while OEG-bis-Pmoc showed more frequent interactions at ∼5 Å but few at 4 Å, suggesting a variety of stacking interactions between phenyl rings. These results indicate that OEG-bis-cHex, OEG-bis-NPmoc and OEG-bis-Pmoc engage in hydrophobic interactions between stickers, correlating well with the experimental finding that these coacervates were destabilized upon the addition of 1,6-hexanediol. Together, these findings provided strong support for the idea that both hydrogen bonding and hydrophobic interactions serve as key driving forces for OEG-bis-X coacervate formation. Furthermore, the time evolution of the number of OEG-bis-X molecules within the largest aggregate revealed distinct aggregation dynamics among the three coacervate-forming variants (Figure S27). While OEG-bis-NPmoc and OEG-bis-cHex rapidly formed stable aggregations with minimal fluctuation, OEG-bis-Pmoc exhibited considerable temporal fluctuation in the number of molecules per aggregate. This observation is consistent with the highest water content, the lowest viscosity observed in microrheological experiments, and the highest molecular dynamics observed in FRAP analysis, suggesting that OEG-bis-Pmoc forms more dynamic and hydrated coacervates than the other two variants.

Effects of Sticker Moieties in OEG-bis-X Coacervates for Selective Partitioning of Small Molecule Guests

To identify the physical properties of a coacervate, determining guest molecules partitioned into the dense phase, i.e., the core, is essential. The selective exclusion or preferential uptake of guest molecules directly influences the downstream biochemical reactions that occur within this unique microenvironment. We evaluated the encapsulation efficiency of different fluorescent dyes [fluorescein, Nile red, tetramethylrhodamine ethyl ester (TMRE), rhodamine B (Rho B), thioflavin T (ThT), and 9-(2,2-dicyanovinyl)­julolidine (DCVJ)] within each OEG-bis-X coacervate by comparing their fluorescence intensities with those of the dilute phase of each dye without coacervates (Figure a). In addition, we observed the fluorescence of each dye within the coacervates by using fluorescence microscopy (Figure b–s). The uptake of hydrophilic fluorescein inside the coacervates appeared to be suppressed in all OEG-bis-X coacervates (Figure a–d). In contrast, all OEG-bis-X coacervates efficiently concentrated hydrophobic Nile red with over 90% encapsulation efficiencies (Figure a). These results suggest that the core of the OEG-bis-X coacervates was relatively hydrophobic compared to the dilute phase. To further elucidate the relationship between the sticker structure and guest uptake, we categorized the stickers based on their chemical features. We first focused on the effect of introducing a nitro group onto the aromatic ring: OEG-bis-NPmoc vs OEG-bis-Pmoc coacervates. NPmoc, bearing an electron-withdrawing nitro group, is expected to enhance π–π interactions with aromatic guest molecules. This may account for the higher encapsulation efficiencies of aromatic guests, such as TMRE, Rho B, ThT, and DCVJ in OEG-bis-NPmoc coacervates (Figure a). In the case of Nile red, ThT, and DCVJ, fluorescence microscopy revealed significantly lower fluorescence intensities in OEG-bis-NPmoc coacervates compared with OEG-bis-Pmoc coacervates, which may be due to NPmoc acting as a quencher (Figure e,f,n,o,q,r). Next, to examine the role of aromaticity, we compared the aromatic OEG-bis-Pmoc with the aliphatic OEG-bis-cHex. While the encapsulation efficiencies of TMRE, Rho B, ThT, and DCVJ were comparable between these two coacervates (Figure a), fluorescence microscopy revealed marked differences in the fluorescence intensity of molecular rotors ThT and DCVJ. ThT showed significantly higher fluorescence in OEG-bis-Pmoc coacervates than in OEG-bis-cHex coacervates (Figure o,p), while DCVJ showed similarly high fluorescence in both coacervates (Figure r,s). This discrepancy may arise from differences in the fluorescence activation mechanisms of the two dyes. ThT typically shows enhanced fluorescence upon binding to ordered hydrophobic regions formed by π–π interactions such as β-sheet-like structures, whereas DCVJ responds more broadly to microviscosity and can show enhanced fluorescence even in isotropic viscous environments such as glycerol. Collectively, these findings highlight that subtle changes in sticker chemistry significantly influence not only guest uptake but also the local molecular environment sensed by different fluorescent probes.

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Partitioning of guest molecules in OEG-bis-X coacervates. (a) Guest encapsulation efficiency (%) of the coacervates. Statistical significance is determined by two-tailed unpaired Student’s t tests (***P < 0.001, **P < 0.01, *P < 0.05, and ns: nonsignificant). Representative CLSM images of OEG-bis-X coacervates incubated with (b–d) fluorescein, (e–g) Nile red, (h–j) TMRE, (k–m) Rho B, (n–p) ThT, and (q–s) DCVJ. [OEG-bis-X] = 4.2 mM and [guest molecule] = 5.0 μM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. Scale bar: 10 μm.

Reduction Responsiveness of OEG-bis-NPmoc CoacervatesDisassembly of OEG-bis-NPmoc Coacervates by Reduction Stimuli

We evaluated the reduction responsiveness of an NPmoc group-containing molecule (OEG-bis-NPmoc) capable of constructing coacervates. As previously reported, chemical and enzymatic reductants can induce multielectron reduction in NPmoc group, even under aqueous conditions, followed by releasing a quinoneimine methide fragment via irreversible 1,6-elimination, as depicted in Figure a. Hence, we prepared OEG-bis-NPmoc coacervates encapsulating 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindolecarbocyanine iodide (DiD; Figure b) or doxorubicin hydrochloride (Figure S28) as guest molecules. The coacervates disappeared within 20 min after adding an aqueous Na2S2O4 (2.5 M, 7.5 μL; 30 equiv against OEG-bis-NPmoc) as a chemical reductant for the NPmoc group into aqueous mixtures containing OEG-bis-NPmoc coacervates (4.3 mM, 150 μL), even though small aggregates remained after 20 min (Figure b and Movie S1). The release ratio of DiD from the coacervates by Na2S2O4 (30 equiv, after 20 min) was estimated to be 64 ± 8.9%. In contrast, the addition of tris­(2-carboxyethyl)­phosphine hydrochloride (TCEP; 1.0 equiv against OEG-bis-NPmoc) at a concentration sufficient to induce the cleavage of the assumed disulfide bond did not induce the disassembly of the OEG-bis-NPmoc coacervates and resulted in a much lower release ratio (4.2 ± 0.6%, Figures b and S28a). Furthermore, in the presence of Na2S2O4, the turbidity of the solution containing OEG-bis-NPmoc coacervates decreased over time compared to that in the presence of TCEP (Figure c,d). The reduction-responsive reaction of OEG-bis-NPmoc induced by Na2S2O4 was analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy (Figure S29), whose samples were prepared after dissolution in a DMSO-d 6-D2O (350 μL/300 μL) mixture (see the Supporting Information for details). The signals assignable to the nitrophenyl group (“a” and “b” protons, Figure S29) almost completely disappeared 30 min after adding Na2S2O4, and multiple new peaks assignable to (hydroxyl)­aminophenyl groups (6.8–7.6 ppm) appeared concurrently while all the signals are not fully assigned yet. In contrast, almost no spectral changes were observed upon the addition of TCEP. This selectivity was consistent with previous reports on NPmoc-containing molecules. , Collectively, these results indicate that the Na2S2O4-induced, reduction-responsive disassembly of OEG-bis-NPmoc coacervate correlates with the chemical reduction of the nitro group and demonstrates a reduction-responsive release of encapsulated guest molecules from OEG-bis-NPmoc coacervates. The molecular design in which such a self-immolative carbamate linkage is used between sticker and spacer offers the possibility to develop other stimuli-responsive coacervates.

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Reduction-stimulus responsiveness of OEG-bis-NPmoc coacervates. (a) Plausible scheme of the reduction reaction of OEG-bis-NPmoc induced by Na2S2O4 and schematic of the reduction-responsive disassembly of OEG-bis-NPmoc coacervates. (b) Representative CLSM images of the OEG-bis-NPmoc coacervates before and after the addition of (i) Na2S2O4 (20 min) and (ii) TCEP (20 min) at ambient temperature. [OEG-bis-NPmoc] = 4.2 mM and [DiD] = 5.0 μM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1). (i) 2.5 M Na2S2O4 aqueous solution (7.5 μL, 30 equiv against OEG-bis-NPmoc) or (ii) 84 mM TCEP aqueous solution (7.5 μL, 1.0 equiv against OEG-bis-NPmoc) was added to the coacervates solution (150 μL). Scale bar: 10 μm. (c) Turbidity (OD600) of solutions containing OEG-bis-NPmoc coacervates (200 μL) before and after the addition of 2.5 M Na2S2O4 (10 μL, 30 equiv against OEG-bis-NPmoc, red plots), 84 mM TCEP (10 μL, 1.0 equiv against OEG-bis-NPmoc, blue plots), or Milli-Q (10 μL, gray plots). [OEG-bis-NPmoc] = 4.3 mM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1). Error bars indicate the SD of the average OD600 (N = 3). (d) Photographs of solutions containing OEG-bis-NPmoc coacervate 80 min after the addition of each solution in a microplate.

Effects of Sticker Moieties in OEG-bis-X Coacervates for the Selective Localization of Macromolecule Guests

We have demonstrated the selective partitioning of small guest molecules into OEG-bis-X coacervates, as shown in Figure . Among the aromatic sticker-bearing coacervates, OEG-bis-NPmoc exhibited stronger π–π stacking than did OEG-bis-Pmoc, as supported by AAMD simulations (Figure S25), leading to enhanced interactions with aromatic guest molecules. However, this also resulted in significant fluorescence quenching for environmentally sensitive fluorescent probes due to the NPmoc group (Figure e,n,q). In contrast, when comparing Pmoc with the aliphatic cHex sticker, OEG-bis-Pmoc coacervates showed a higher uptake of aromatic guest molecules (Figure a). Nevertheless, for certain guest probes such as viscosity-sensitive DCVJ, which reports on local microviscosity, both the OEG-bis-Pmoc and OEG-bis-cHex coacervates exhibited comparable uptake and fluorescence intensity, possibly reflecting some shared similar physicochemical environments within the coacervates.

Motivated by these findings, we further compared the partitioning behavior of macromolecular guests in the OEG-bis-Pmoc and OEG-bis-cHex coacervates (representing simple aromatic and aliphatic sticker types, respectively). Previous studies have reported that guest molecular size limits partitioning into biomolecular condensates. , Thus, we examined the uptake of tetramethylrhodamine-labeled dextran (TMR-dextran), which varied depending on the molecular weight of dextran (average M w: 4,400, 65,000–85,000, and 155,000 g mol–1), as macromolecule guests using CLSM (Figure ). The smallest TMR-dextran (M w: 4,400 g mol–1) was concentrated inside both the OEG-bis-Pmoc and OEG-bis-cHex coacervates (Figure a,b). In the case of the middle TMR-dextran (M w: 65,000–85,000 g mol–1), accumulation at the interface of OEG-bis-cHex coacervates and uptake by OEG-bis-Pmoc coacervates was observed (Figure c,d). The enhanced accessibility of TMR-dextran into OEG-bis-Pmoc coacervates may be attributed to their higher water content and fluidity, which facilitate the uptake of hydrophilic polymers bearing hydrophobic anchors such as TMR. The largest TMR-dextran (M w: 155,000 g mol–1) accumulated at the interface of both the OEG-bis-Pmoc and OEG-bis-cHex coacervates (Figure e,f). In previous reports of intrinsically disordered protein-based droplets (LAF-1, WHI3, and NPM1), TMR-dextran with a molecular weight of ∼70,000 g mol–1 is excluded from the droplet interior. This exclusion could be attributed to the densely packed internal network of these protein condensates. In contrast, in our system, the middle and largest TMR-dextran were not completely excluded but rather accumulated at the interface, suggesting a mechanism different from that of classical size exclusion. Qiao et al. recently reported dextran-layer-bounded coacervates using dextran with a higher M w. , Their complex coacervates, composed of protamine sulfate and folic acid, could induce a possible interfacial interaction between dextran without TMR and the coacervates. Thus, the mechanisms of interfacial accumulation were significantly different from those in our system. As supporting evidence, we demonstrated that another low-M w guest (i.e., DCVJ) with its relatively high concentration could remove the TMR-dextran layers from the coacervate surfaces (Figure S30), indicating that the TMR-anchor contributes to dextran accumulation most probably through hydrophobic interactions. This guest-competition-induced release of dextran layers represents a new approach for modulating coacervate interfaces.

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Differences in the localization behaviors of TMR-dextran and its dynamics at the surface of OEG-bis-X coacervates. Representative CLSM images and line plots of OEG-bis-X coacervates incubated with TMR-dextran with M w: (a,b) ∼4,400, (c,d) ∼65,000–85,000, and (e,f) ∼155,000 g mol–1. Representative TIRFM images and histograms of dwell times (N = 50 spots) for the middle TMR-dextran are at the surface of (g,h) OEG-bis-Pmoc and (i,j) OEG-bis-cHex. [OEG-bis-X] = 4.2 mM and [TMR-dextran (M w ≈ 65,000–85,000 g mol–1)] = 5.0 nM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. Scale bar: 5 μm. (k) Histograms showing the distributions of diffusion coefficients of single TMR-dextran molecules on the surface of OEG-bis-X coacervates (orange: OEG-bis-cHex, blue: OEG-bis-Pmoc), calculated from the MSD slopes within a 6 ms time window. The region corresponding to diffusion coefficients below 0.015 μm2 s–1 (highlighted in yellow) represents the immobile population, which was determined using glass-immobilized TMR molecules under the same imaging conditions. Statistical analyses were performed using the Mann–Whitney U test. [OEG-bis-X] = 4.2 mM and [TMR-dextran (M w ≈ 65,000–85,000 g mol–1)] = 5.0 nM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. (l) Number of intramolecular (intra.) or intermolecular (inter.) hydrogen bonds between OEG-bis-X molecules analyzed by AAMD simulations. (m) RDF of carbon atom in the sticker (sticker-C) or oxygen atom in the spacer (spacer-O) within OEG-bis-X molecules inside OEG-bis-X (X = Pmoc: left panel, cHex: right panel) coacervate models. The interior of the coacervate model is highlighted in yellow. The reference center is defined by the center of mass of aggregated OEG-bis-X molecules.

Investigation of Structural Heterogeneity within OEG-bis-X Coacervates

Although both coacervates exhibited similar surface accumulation of large TMR-dextran, we observed a clear difference in the distribution of the middle TMR-dextran between OEG-bis-Pmoc and OEG-bis-cHex coacervates. This contrast suggested that the molecular-level sticker structure may play a more pronounced role in modulating the accumulation behavior of the middle TMR-dextran. To further investigate the enhanced surface accumulation of the middle TMR-dextran in OEG-bis-cHex coacervates compared with OEG-bis-Pmoc, we employed TIRFM to analyze the dynamics of the middle TMR-dextran molecules on the coacervate surfaces (Figures g–k and S31). Most of the TMR-dextran molecules exhibited short-lived trajectories on OEG-bis-Pmoc coacervates, with a half-time (t 1/2) of 49.6 ms (Figure g,h). In contrast, many molecules showed long-lived, restricted trajectories on OEG-bis-cHex coacervates, with t 1/2 values of 165.9 ms (Figure i,j). The calculated dissociation rate constants (k off) for TMR-dextran molecules on OEG-bis-Pmoc and OEG-bis-cHex coacervates were 13.97 and 4.18 s–1, respectively, indicating significantly slower dissociation dynamics on OEG-bis-cHex coacervates. To separately evaluate the lateral mobility, we analyzed only the fluorescent spots that exhibited measurable two-dimensional (2D) diffusion on the coacervate surfaces (Figure k). The median diffusion coefficients differed significantly between the two coacervates (p < 0.0001, two-sided Mann–Whitney U test), with OEG-bis-cHex coacervates showing markedly lower D value (0.057 μm2 s–1) than OEG-bis-Pmoc (0.456 μm2 s–1). Notably, the overall diffusion of TMR-dextran molecules was extremely slow, and diffusion coefficients below 0.015 μm2 s–1 were thus classified as immobile based on the diffusion coefficient determined for glass-immobilized TMR molecules under the same imaging conditions. Using this threshold, approximately 14% of TMR-dextran molecules on OEG-bis-cHex coacervates were categorized as immobile, compared with approximately 5% for OEG-bis-Pmoc coacervates, demonstrating stronger restriction of dynamics and greater surface heterogeneity on the OEG-bis-cHex coacervate surface. Furthermore, the number of TMR-dextran molecules bound on the OEG-bis-cHex coacervate surface was approximately three times higher than for OEG-bis-Pmoc coacervates, as observed in the TIRF images (Figure S31b,c). This result is also consistent with the slower dissociation rate (k off = 4.18 s–1) and longer dwell time (t 1/2 = 166 ms) of TMR-dextran on OEG-bis-cHex. These results suggest that the coacervate surface formed by OEG-bis-cHex provides a more favorable environment for the surface accumulation of TMR-dextran, potentially due to slower and more restricted surface dynamics compared with that of OEG-bis-Pmoc.

Next, a detailed analysis of the AAMD simulations were conducted to gain further insights into the structural differences within OEG-bis-X coacervates. Based on CLSM and TIRFM results, we hypothesized that the surface of OEG-bis-cHex coacervates develop more hydrophobic regions compared with OEG-bis-Pmoc coacervates, resulting in greater structural heterogeneity. To assess this hypothesis, we examined the structures within OEG-bis-Pmoc and OEG-bis-cHex coacervates using the results of the AAMD simulations. The AAMD simulations were based on an aggregate model with a radius of 23–25 Å as a representative coacervate model. We analyzed the number of hydrogen bonds formed between the OEG-bis-X molecules. The results showed that intramolecular hydrogen bonds were minimal for both OEG-bis-Pmoc and OEG-bis-cHex, with no significant time-dependent changes (Figures k and S32). In contrast, intermolecular hydrogen bonds were more numerous than intramolecular bonds, especially in OEG-bis-cHex. These results suggest that OEG-bis-cHex has a higher tendency to form subclusters in the aggregates compared with OEG-bis-Pmoc. Next, we analyzed the spatial distribution of water molecules across the aggregates using the radial distribution function (RDF), as shown in Figure S33. In OEG-bis-Pmoc, the water distribution gradually increased from the center to the surface. However, in OEG-bis-cHex, the water density was relatively low at the core as well as ∼4 and ∼20 Å from the center, indicating a more heterogeneous water distribution compared with that of OEG-bis-Pmoc. We also examined the radial distribution of OEG-bis-X molecules by tracking the central oxygen atom in the spacer unit (spacer-O) and the carbon atom in the sticker unit (sticker-C) (Figure l). In the OEG-bis-Pmoc coacervate model, the distributions of spacer-O and sticker-C were almost identical and spread uniformly. In the OEG-bis-cHex coacervate model, the distribution of spacer-O and water molecules were consistent, whereas the distribution of the spacer-O and sticker-C clearly diverged, especially around the coacervate surface (∼18 Å), revealing a structure in which hydrophobic and hydrophilic regions are separated (Figures l and S33). These results indicate that OEG-bis-Pmoc coacervates maintain a dynamic structure stabilized by multiple intermolecular interactions with continuous molecular exchange. In contrast, OEG-bis-cHex coacervates exhibit structural heterogeneity, particularly at the surface, where hydrophobic regions are generated. Such structural features may contribute to the surface accumulation of TMR-dextran molecules and the differences in the water content and viscosity.

Interfacial Stabilization and Multicompartmental Assembly Enabled by TMR-Dextran

If the coacervate surface is covered with TMR-dextran, fusion events between coacervates are expected to be suppressed, resulting in a stabilization of the coacervate size. Here, we assessed coacervate growth over time with or without TMR-dextran using dynamic light scattering (DLS) analysis (Figure a). In the absence of TMR-dextran, the size of the OEG-bis-cHex coacervate clearly increased within 60 min. In contrast, the change in the size of the coacervates was significantly suppressed in the presence of TMR-dextran, suggesting that coalescence was inhibited (Figure b). DLS analysis also revealed that the presence of TMRE or dextran without TMR did not suppress coacervate growth. These results are consistent with the mechanism described above in which the interactions between TMR appended to dextran and OEG-bis-cHex are crucial for the interfacial accumulation of TMR-dextran on the coacervates (Figure c).

5.

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TMR-dextran coating suppresses the fusion of OEG-bis-cHex coacervates, resulting in a multicompartmental assembly by centrifugation. The stability of the OEG-bis-cHex coacervates was evaluated by measuring the coacervate size using DLS. (a) Schematic of sample preparation for DLS measurements. (b) Change in the size of OEG-bis-cHex coacervate over time. [OEG-bis-cHex] = 4.2 mM and [TMR-dextran L (M w ≈ 155,000 g mol–1), Dextran L (M w ≈ 150,000 g mol–1), or TMRE] = 5.0 μM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. (c) Schematic of the interfacial accumulation of TMR-dextran on OEG-bis-cHex coacervates. Core–shell structures can be constructed by the sequential addition of two guest molecules (DCVJ and TMR-dextran) for the OEG-bis-cHex coacervate. (d) Representative CLSM image and line plot of the core–shell OEG-bis-cHex coacervate. Scale bar: 5 μm. Representative (e) 2D and (f) 3D CLSM images of the core–shell OEG-bis-cHex coacervate superstructures constructed by centrifugation (4,000×g, 2 min). [OEG-bis-cHex] = 4.2 mM, [DCVJ] = 0.1 μM, and [TMR-dextran (M w ≈ 150,000 g mol–1)] = 5.0 μM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. Scale bar: 30 μm. Representative CLSM images of (g) pair A (DCVJ/TMR-dextran and mitoxantrone/TMR-dextran) and (h) pair B (DiO/TMR-dextran and DiD/TMR-dextran) core–shell coacervates. [OEG-bis-cHex] = 4.2 mM, [DCVJ or mitoxantrone] = 0.1 μM, [DiO or DiD] = 5.0 nM, and [TMR-dextran (M w ≈ 150,000 g mol–1)] = 1.4 μM for pair A or 2.4 μM for pair B in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1) at ambient temperature. Scale bar: 10 μm.

Core–shell structures can be constructed by the sequential addition of two guest molecules with different affinities. For the OEG-bis-cHex coacervate, adding DCVJ first followed by TMR-dextran resulted in a core–shell structure (Figure d). Notably, similar core–shell structures were also obtained when TMR-dextran was added prior to DCVJ, suggesting that the TMR-dextran shell is semipermeable (Figure S34). Furthermore, the core–shell coacervates formed loosely packed superstructures upon centrifugation (4,000×g, 2 min, Figure e,f). Within these assemblies, individual coacervates remained dynamic, particularly at the periphery (Figure S35).

To further explore the construction of multicompartmental architectures, we mixed two types of core–shell coacervates labeled with distinct fluorescent dyes (pair A: DCVJ/TMR-dextran and mitoxantrone/TMR-dextran core–shell coacervates; pair B: 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO)/TMR-dextran and DiD/TMR-dextran core–shell coacervates). After mixing for pair A, CLSM observations revealed that both DCVJ and mitoxantrone fluorescence appeared in nearly all coacervates, indicating pronounced intermixing of guest molecules between different coacervate cores, even without their fusion (Figure g). In contrast, pair B remained largely independent (Figure h). Although both pairs A and B possess the same semipermeable TMR-dextran shells, the extent of intermixing strongly depended on the partitioning characteristics of the guest molecules. In pair A, relatively hydrophilic and small guests like DCVJ and mitoxantrone underwent more active exchange, leading to noticeable intermixing of core fluorescence. In contrast, the highly hydrophobic DiO and DiD bearing long alkyl chains in pair B were more strongly retained within the cores and became kinetically trapped, thereby showing limited intermixing under the same conditions. Nonetheless, upon centrifugation (4,000×g, 2 min), packed pair B coacervates occasionally exhibited partial intermixing, indicating that passive molecular exchange was promoted under external compression, which resulted in partial intermixing even in pair B (Figure S36). Collectively, these results suggest that by carefully selecting guest molecules based on their partitioning tendencies, multicompartmental architectures can be rationally constructed while maintaining coacervate structural integrity.

Conclusions

We successfully developed a series of simple sticker-spacer-type molecules, OEG-bis-X (X = NPmoc, Pmoc, cHex), capable of forming liquid-like coacervates by LLPS. By modifying the sticker motifs of OEG-bis-X, we identified the sticker motifs required for coacervate formation. We evaluated the selective partitioning of various small molecule guests into these coacervates. All three types of coacervates exhibit hydrophobic microenvironments that sequester hydrophobic guest molecules while excluding hydrophilic guests. We also demonstrated the reduction-responsive degradation of OEG-bis-NPmoc coacervates, which resulted in the release of the encapsulated guests. Notably, subtle differences in the sticker groups significantly influenced the water content and viscosity as well as the uptake efficiency of cationic and zwitterionic dyes. This suggests that the sticker motifs not only affect guest partitioning but also modulate the internal properties of the coacervates such as hydrophobicity or structural heterogeneity. Intriguingly, accumulation of macromolecules (i.e., zwitterionic dye-labeled polysaccharides) at the interface of the coacervates is dependent on the sticker motif. TIRFM imaging and AAMD simulations revealed that OEG-bis-cHex coacervates exhibited structural heterogeneity with hydrophobic regions at the surface. The interfacial accumulation of macromolecules enhanced the stability of the coacervates against an uncontrollable size expansion due to coalescence. Most previous studies on the resistance to coacervate coalescence were related to complex coacervates but not simple coacervates. ,, In this study, a new method was developed to cover simple coacervates with macromolecules, where the synergetic combination of weak hydrophobic interactions between OEG-bis-cHex and macromolecules and size exclusion effects play important roles. Further research on the dependence of sticker or spacer parts on stimuli responsiveness and guest-selective sequestration is in progress in our laboratory, as well as the detailed elucidation of the internal microenvironments of coacervates. Our findings contribute to the rational molecular design of coacervates for tailored applications in the fields of materials science and biotechnology.

Supplementary Material

au5c01238_si_001.pdf (15.8MB, pdf)
Download video file (4.6MB, avi)

Acknowledgments

This work was supported in part by JSPS KAKENHI (24H01126 and 25K18078 awarded to S.L.H., and 23H01815 awarded to M.I.), Tokai Pathways to Global Excellence (T-GEx), the MEXT Strategic Professional Development Program for Young Researchers (S.L.H.), Toyota Riken Research Grants (S.L.H. and M.I.), and the Inoue Foundation (S.L.H.). The authors thank Drs. Takahiro Fujiwara and Akihiro Kusumi for developing the analysis software used for single-molecule imaging. The authors thank Editage (www.editage.jp) for English language review.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01238.

  • Full experimental and characterization details, 1H and 13C NMR spectra for all compounds, fluorescence microscopy images, and AAMD simulation results (PDF)

  • Disassembly of OEG-bis-NPmoc coacervates by reduction stimuli (AVI)

S.L.H.: Conceptualization, methodology, validation, formal analysis, investigation, and writingoriginal draft. K.M.H.: Methodology, validation, formal analysis, and writingreview and editing. R.F.: Methodology and validation. K.K.: Methodology, validation, and formal analysis. N.Y.: Methodology, validation, formal analysis, and writingreview and editing. K.G.N.S.: Writingreview and editing. M.I.: Conceptualization, methodology, validation, formal analysis, investigation, writingreview and editing, and supervision. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

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