Introduction

Coacervation is the process of phase separation under aqueous conditions to yield condensed liquid-like droplets with micrometer sizes [1]. Coacervates with a spherical morphology at the micrometer scale have been frequently obtained from charged biopolymers such as gelatin under aqueous conditions [2]. Recently, the biomolecular condensates inside living cells have been revealed to be liquid-like droplets that are obtained via liquid‒liquid phase separation of biomolecules [3, 4]. Recent studies have shown that biomolecular condensates composed of proteins and nucleic acids lack surrounding membranes and are involved in essential processes of living systems, including signal transduction, metabolism, and gene regulation [5, 6]. In contrast, artificial condensates comprising low-molecular-weight compounds [7,8,9] have garnered comparatively less attention than (bio)polymers. Nevertheless, artificial condensates constructed from such simpler molecular components could deepen the understanding of the distinct chemical and physical principles underlying the behavior of more complex biomolecular condensates as simplified models and also provide potentially useful and cost-effective soft materials for (bio)applications [10].

In host‒guest chemistry research, linear oligoethylene glycol (OEG) derivatives bearing aromatic groups at both ends have been called podands, which can exhibit an affinity for cations [11]. On the other hand, recent studies revealed that simple OEG derivatives bearing aromatic dipeptides [12] as well as polycyclic aromatic groups (i.e., pyrene) [10] at both ends could yield coacervates under aqueous conditions. In addition, we recently reported that several OEG derivatives bearing appropriate hydrophobic groups, such as phenyl groups or cyclohexyl groups, could also self-condensate into coacervates under aqueous conditions [13]. These findings reveal that the OEG moiety can be regarded as an appropriate spacer for the molecular design of minimal sticker-and-spacer motifs within phase-separating proteins [14]. In this study, we extend the molecular design further from the previous amide-linked OEG derivatives [12, 13] to thioether-linked OEG derivatives [10]. We expect that the built-in thioether linkage, i.e., the sulfide group, allows for oxidation-responsiveness, as illustrated in Fig. 1A. Indeed, we [15, 16] and other research groups [17, 18] have recently explored self-assembling small molecules bearing sulfide groups to obtain oxidation-responsive hydrogels. Furthermore, a sulfide group present in the side chains of methionine in proteins is sensitive to oxidation and evidently related to essential biological functions [19, 20]. However, oxidation-responsive artificial condensates composed of low-molecular-weight compounds are still relatively unexplored. Here, we report that a thioether-linked OEG derivative self-condensates into liquid-like droplets capable of oxidation-responsive disassembly under aqueous conditions (Fig. 1B).

Fig. 1
figure 1

Construction of oxidation-responsive coacervates composed of OEG(BzlS)2. A Chemical structure of OEG(BzlS)2 as a minimal sticker-and-spacer motif and its oxidation-induced transformation into OEG(BzlSO)2. B Schematic illustration (not to scale) of the formation of coacervates as liquid-like droplets (artificial condensates) composed of OEG(BzlS)2 and its oxidation-responsive disassembly associated with the molecular transformation from sulfide to sulfoxide shown in (A)

Full size image

Materials and methods

Experimental generals

Unless stated otherwise, all commercial reagents were used as received, and the water used in the experiments was ultrapure water obtained from a Millipore system with a specific resistance of 18 MΩ•cm. The synthesis and characterization of OEG(BzlS)2 and OEG(BzlSO)2 and further experimental details are provided in the Supplementary Information.

Preparation of coacervates

Typically, a dimethyl sulfoxide (DMSO) solution of OEG(BzlS)2 (100 mM, 20 µL) was added to aqueous phosphate buffer (100 mM, pH 7.5, 180 µL), and the mixture was vortexed immediately for ~3 s. The resultant dispersion {[OEG(BzlS)2] = 10 mM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1), 20 µL} prepared in a glass vial was spotted on a glass coverslip (diameter: 25 mm, Paul Marienfeld) and subjected to microscopic observation. The details of the observation settings and conditions can be found in the Supplementary Information. Additionally, the resulting dispersion was transferred into a plastic 96-well microplate to measure turbidity using an Infinite 200 PRO microplate reader (Tecan, Switzerland). Further details are provided in the Supplementary Information.

Results and discussion

Coacervation, which results in the formation of artificial condensates, can be agilely characterized by measuring turbidity. Indeed, OEG(BzlS)2 showed high DMSO solubility, resulting in the formation of transparent DMSO solutions (Fig. 2A_i) at even high concentrations (e.g., 50 mg/mL), whereas mixing the DMSO solution with an aqueous buffer such as phosphate buffer (pH 7.5) {typically, 100 mM phosphate buffer (pH 7.5)/DMSO (/v = 9:1): standard conditions in this study} resulted in a cloudy dispersion immediately, as shown in Fig. 2A_ii. Concentration-dependent turbidity was evaluated by measuring the absorbance at 600 nm, and the results are summarized in Fig. 2B, indicating that OEG(BzlS)2 can yield coacervates above ~2 mM. The turbidity decreased and became saturated within ~6 h (vide infra), which may relate to sedimentation, as shown by the corresponding micrographic images (Supplementary information Fig. S5), and centrifugation indeed facilitated bulk phase separation [12] (Supplementary information Fig. S6). Additionally, we found that similar OEG(BzlS)2 coacervates can be prepared by using an ethanol solution instead of DMSO but less effectively with acetonitrile (Supplementary information Fig. S7). Furthermore, mixing OEG(BzlS)2 (without dissolution in DMSO) with aqueous buffer {100 mM phosphate buffer (pH 7.5)} did not result in a cloudy dispersion even after ultrasonication. However, heat-and-cool treatment of the aqueous mixture resulted in a cloudy dispersion in which coacervates were found (Supplementary information Fig. S7). For practicality, the DMSO stock solution protocol was used in the following study. In sharp contrast to OEG(BzlS)2, the chemically oxidized OEG(BzlSO)2 showed higher aqueous solubility; thus, a transparent aqueous solution was obtained even at high concentrations, such as 10 mM, as shown in Fig. 2A_iii. In this study, OEG(BzlSO)2, which was obtained as a mixture of three possible isomers resulting from the chirality of the sulfoxide groups, was used because of the difficulty in finding the appropriate conditions for the separation of each isomer. Notably, recent research by another group [10] indicated that a similar phenyl group-terminated OEG derivative bearing additional carbonyl groups between the phenyl groups and the central OEG moiety exhibited no coacervation ability but formed aggregates; i.e., the absence of the carbonyl groups in OEG(BzlS)2 was critical for coacervate formation. The present findings thus highlight the importance of ensuring the balance of sticker hydrophobicity—that is, its self-association ability—and linker hydrophilicity to accommodate water molecules. To improve the molecular design of OEG derivatives for the development of coacervates, further research is needed to investigate the influence of each motif.

Fig. 2
figure 2

Formation of OEG(BzlS)2 coacervates. A Photographs of (i) a DMSO solution of OEG(BzlS)2, (ii) an aqueous dispersion containing OEG(BzlS)2, and (iii) an aqueous solution of OEG(BzlSO)2 in glass vials with 200 µL inserts. B Concentration-dependent turbidity (absorbance at 600 nm) of OEG(BzlS)2 and OEG(BzlSO)2. C Representative microscopy images (DIC) of OEG(BzlS)2 coacervates. D Time-lapse microscopy images of OEG(BzlS)2 coacervates showing a coalescence event (a magnified part of Supplementary Movie S1; see also Supplementary information Fig. S8 for whole images at selected times). Conditions: [OEG(BzlS)2] = 10 mM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1), ambient temperature. Scale bar: 30 µm for (C) and 10 µm for (D)

Full size image

To obtain insights into the morphology of the coacervates of OEG(BzlS)2, optical microscopic observations were carried out. As shown in Fig. 2C, micrographic image analysis revealed the formation of spherical droplets with a typical size of several µm immediately after sample preparation by mixing a DMSO solution with aqueous buffer. In addition, the coalescence of the generated droplets as well as their Ostwald ripening (Fig. 2D) and wetting on the glass surface were observed (Supplementary Movie S1, Supplementary Information Fig. S8), which are consistent with liquid-like properties [1]. Furthermore, the water content in OEG(BzlS)2 coacervates was evaluated to be ~45% according to experiments based on weight measurements [12], which is within the range of typical liquid droplets (>~40%) [1]. Collectively, these data demonstrated that OEG(BzlS)2 self-condensates into liquid-like coacervates under aqueous conditions.

To evaluate the substance encapsulation propensity of the OEG(BzlS)2 coacervates, confocal laser scanning microscopy (CLSM) observations were carried out after the addition of typical fluorescent dyes (Fig. 3A). As shown in Fig. 3A_i, Nile red, a representative hydrophobic dye, was effectively encapsulated in the coacervates, whereas rather hydrophilic fluorescein was excluded from the coacervates (Fig. 3A_iv). Nevertheless, other hydrophobic dyes, such as 9-(2,2-dicyanovinyl)julolidine (DCVJ) (Fig. 3A_ii) and fluorescein-(allyl)2 (Fig. 3A_iii) [21], which are less hydrophilic fluorescein derivatives, were sequestered inside the coacervates. This partitioning behavior, which depends mainly on the hydrophobic properties of dyes, is almost comparable to that of coacervates composed of amide-linked OEG derivatives [13]. Moreover, the encapsulation of the fluorescein-based dye straightforwardly allowed the study of molecular dynamics inside the OEG(BzlS)2 coacervates by means of the fluorescence recovery after photobleaching (FRAP) technique, which is commonly utilized for investigating the dynamics of biomolecular condensates [22]. As shown in Fig. 3B, the fluorescence recovery was substantiated within ~60 s, with a plateau ( ~ 98% recovery). The obtained FRAP data were analyzed with a simple exponential model (Fig. 3B_ii), which provided a t1/2 (the time at which half of the fluorescence recovered) of 15 s (n = 3). This result is consistent with the formation of liquid-like coacervates [10, 12, 13] but not solid-like coacervates.

Fig. 3
figure 3

Partitioning of the fluorescent guest molecules in OEG(BzlS)2 coacervates. A Representative CLSM images of the following fluorescent dyes: (i) Nile red, (ii) DCVJ, (iii) fluorescein-(allyl)2 [21], and (iv) fluorescein. B Fluorescence recovery after photobleaching (FRAP) experimental results with fluorescein-(allyl)2 (i: Representative time-lapse images; the white broken circle in the prebleach image shows the photobleach area; ii: Fluorescence recovery profile; n = 3). Conditions: [OEG(BzlS)2] = 10 mM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1), [fluorescent dyes] = 5 µM, ambient temperature. Scale bar: 10 µm for (A) and 5 µm for (B_i)

Full size image

With liquid-like coacervates composed of a single molecular component bearing a sulfide group, i.e., OEG(BzlS)2, we next sought to investigate the oxidation-responsive propensities of these coacervates. The sulfide group can be oxidized into sulfoxide and subsequently sulfone, depending on the oxidation conditions [23]. According to previous studies, including ours [15,16,17,18], on self-assembling molecules bearing a sulfide group, hydrogen peroxide (H2O2), which can act as a relatively mild oxidant under aqueous conditions depending on the concentration, can convert sulfide to sulfoxide with only a small amount of sulfone generation.

When aqueous H2O2 (9.7 M, 150 µL) was added to the dispersion containing OEG(BzlS)2 coacervates (5.0 mM, 200 µL), the absorbance value at 600 nm (turbidity) decreased significantly from 1.6 to 0.14 at 6 h after addition (Fig. 4A). In contrast, the addition of the same amount of water (150 µL) without H2O2, which is comparable to the simple dilution from 5.0 mM to 2.9 mM OEG(BzlS)2 (still above the critical concentration of ~ 2 mM, Fig. 2B), did not induce such a substantial decrease. Nevertheless, a time-dependent decrease in turbidity was observed after the addition of water without H2O2, which could be attributed to the coalescence of droplets discussed above and concurrent sedimentation. In accordance with the decrease in turbidity induced by H2O2, the number of droplets observed by optical microscopy decreased noticeably within 1 h after the addition of H2O2 (Fig. 4B).

Fig. 4
figure 4

Oxidation-responsiveness of OEG(BzlS)2 coacervates. A Time-dependent turbidity (n = 3) of a dispersion containing OEG(BzlS)2 coacervates after the addition of (i) water without H2O2 and (ii) aqueous H2O2. The corresponding photographs of the microplate are shown. B Representative time-lapse microscopy images of OEG(BzlS)2 coacervates after the addition of (i) water without H2O2 or (ii) aqueous H2O2. C Representative 1H NMR spectra {400 MHz, DMSO-d6/D2O (v/v = 5:1), rt} of OEG(BzlS)2 coacervates 6 h after the addition of (i) water without H2O2 and (ii) aqueous H2O2. The spectra of (iii) OEG(BzlSO)2 and (iv) OEG(BzlS)2 under the same conditions are also shown (several peaks overlap with solvents). The asterisk (*) denotes the peaks of the solvents. Conditions: (before): [OEG(BzlS)2] = 5.0 mM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1), (after): a mixture of solutions A/B (v/v = 4:3); solution A: [OEG(BzlS)2] = 5.0 mM in 100 mM phosphate buffer (pH 7.5)/DMSO (v/v = 9:1), solution B: aqueous H2O2 (9.7 M) or water without H2O2, ambient temperature. Scale bar: 30 µm for (B)

Full size image

To investigate the molecular transformation during the oxidation-responsive disassembly of OEG(BzlS)2 coacervates, 1H NMR spectroscopic measurements were conducted (Fig. 4C). As shown in Fig. 4Cii, one of the peaks assignable to benzyl protons (Ha) was found at 4.12 ppm for the sample 6 h after the addition of H2O2 but not after the addition of water without H2O2 (Fig. 4Ci). Furthermore, the spectrum after the addition of H2O2 is reasonably comparable to that of the chemically synthesized OEG(BzlSO)2 (Fig. 4Ciii). Thus, the conversion from sulfide OEG(BzlS)2 to sulfoxide OEG(BzlSO)2 was estimated to be 63% (i.e., 1.1 mM OEG(BzlS)2 remained) on the basis of the integral value of Ha over that of He and He′ (phenyl group). As discussed above (Fig. 2), OEG(BzlSO)2 did not show coacervate-forming ability in this concentration range (at least below 10 mM), and OEG(BzlS)2 showed attenuated ability at the estimated concentration of 1.1 mM. Collectively, these results indicate that OEG(BzlS)2 coacervates can exhibit H2O2-responsive disassembly, mainly through the conversion of sulfide groups into sulfoxide groups.

Conclusion

We demonstrated that coacervates as liquid-like droplets can be obtained from a simple, linear oligo(ethylene glycol) derivative bearing benzyl sulfide groups at both ends, which can be considered low-molecular-weight compounds (Mw < 500). In addition, we revealed the H2O2-responsive disassembly of the obtained coacervates as a result of the conversion of the sulfide groups to less hydrophobic sulfoxide groups. Selective reduction of the resulting sulfoxide to sulfide is more difficult, especially under aqueous conditions [24, 25], whereas reversible formation of liquid-like droplets should be addressed in future studies. We believe that comprehensive studies on this class of simple coacervates composed of low-molecular-weight compounds [7,8,9,10, 12, 13, 26] could have the potential to broaden the scope for the development of (bio)functional materials with a range of applications [27, 28] as well as the construction of complex and hierarchical self-assembled structures [29] that can emulate living cell systems. Consequently, the present reductionist approach, which employs simplified molecular components to construct coacervates bearing certain similarities to biomolecular condensates, may facilitate our comprehension of the fundamental principles underlying biological systems.