Introduction

Proteins that undergo liquid‒liquid phase separation (LLPS) to form dense droplets (coacervates) have been shown to play important roles in many biological systems, including the regulation of cell function and tissue development [1]. Tropoelastin, the soluble precursor of elastin, is a representative protein that forms coacervates in response to various stimuli, such as temperature and salt concentration [2]. Elastin is an extracellular matrix (ECM) protein found in blood vessels, lungs, ligaments, and skin [3]. The protein organizes into networks of elastic fibers that can support indefinite cycles of stress and relaxation [2,3,4]. LLPS of tropoelastin has long been considered a crucial step in the formation of elastic fibers. After being secreted into the extracellular space, tropoelastin forms coacervates that are deposited on fibrillin, followed by enzyme-mediated intermolecular crosslinking to produce mature elastin.

Tropoelastin is a 60–70 kDa protein comprising highly repetitive amino acid sequences. There are three types of domains in tropoelastin: proline-rich hydrophobic, glycine-rich hydrophobic, and crosslinking domains (Fig. 1) [5, 6]. The proline-rich hydrophobic domains are rich in proline (P) and glycine (G), which are known as structure-breaking residues [7], and are thus intrinsically disordered. On the other hand, glycine-rich hydrophobic domains share the repeating motif Z1GGZ2G, where Z is valine (V) or leucine (L), which tends to form β-sheet structures [8]. In the proline-rich hydrophobic domains, P appears repeatedly at a rate of once every 4–7 residues, whereas in the glycine-rich hydrophobic domains, there is no such characteristic occurrence of P. The crosslinking domains are rich in lysine (K) residues, the primary amines of which are involved in intermolecular crosslinking. Tropoelastin exhibits lower critical solution temperature (LCST)-type phase separation, is soluble at low temperatures, and forms coacervates at high temperatures. This behavior has also been observed in elastin-like polypeptides (ELPs), which are synthetic or genetically engineered proteins with simplified repetitive sequences of tropoelastin [9, 10]. A typical ELP consists of a repeat of VPGXG found in the proline-rich domain of tropoelastin, where X is any amino acid except P. The LCST is fine-tuned by the composition of X and the molecular weight of the ELP [11]. Garcia Quiroz and Chilkoti reported sequence heuristics to encode LCST behavior in ELPs; the LCST is exhibited by enriching the intrinsically disordered region composed of P−Xn–G motifs, where n = 0–4 and X are largely nonpolar [12].

Fig. 1
figure 1

Diagram of human tropoelastin domains and frequently found motifs. Exon 1 (S) represents the signaling peptide, and exon 36 represents the C-terminal domain [5]. The unique hydrophilic domain is encoded by exon 26a [6]. Human tropoelastin lacks exons 34 and 35

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In addition to their fundamental importance in biology, the dynamic nature of ELP coacervates makes them attractive platforms as innovative materials in bioengineering and nanomedicine. For example, ELPs have been utilized as purification tags for recombinant proteins [13] and stimuli-responsive drug delivery vehicles [14, 15]. Inspired by the roles of biological LLPS systems, ELP coacervates have recently been used for the isolation of nucleic acids [16] or reversibly triggerable compartments within artificial cells [17]. Furthermore, ELP coacervates can be formed in living cells to generate artificial membraneless organelles [18,19,20]. Ge et al. expressed a fusion protein consisting of an ELP and green fluorescent protein (GFP) in Escherichia coli cells [18]. As expression increased, a 1.5 μm droplet rich in the fusion protein formed in the cells. The fluid-like behavior of these droplets was characterized by fluorescence recovery after photobleaching (FRAP) experiments. Pastuszka et al. expressed GFP-ELP fusion proteins in HEK-293 human fetal kidney cells [19]. The cytoplasmic diffusion coefficient of GFP-ELP determined from FRAP was 7.4 ± 1.4 μm2s−1 below the transition temperature, similar to that of cytosolic proteins. Above the transition temperature, GFP-ELP formed coacervates 0.1−2 μm in diameter, and their diffusion coefficient decreased to 1.1 ± 0.2 μm2s−1. The assembly and disassembly of the microdomains were rapid, with half-lives of 3.8 and 1.0 min, respectively. Li et al. succeeded in expressing GFP-ELP in individual cells of zebrafish embryos and revealed the reversible temperature responsiveness of the phase-separated structure [20]. These stimuli-responsive microcompartments may allow for the switching of cellular activity in the future and may be useful tools for developmental biology and other biological studies.

Although the phase transition behavior of ELPs with VPGXG repeat sequences is considered reversible, coacervates of synthetic proteins that more closely mimic tropoelastin have been found to undergo an irreversible maturation process, depending on the timescale observed. Vidal Ceballos et al. examined the dynamics of LLPS droplets via a minielastin model that mimicked the alternating domain structure of tropoelastin [21]. The minielastin underwent a temperature-dependent phase transition to droplets; the phase transition was reversible within a few minutes of incubation. In contrast, continued incubation above the transition temperature resulted in irreversible morphological changes in the droplets over time; granular internal structures appeared within the droplets at approximately 60−70 min (Fig. 2A). FRAP and particle-tracking microrheology further demonstrated that the initially homogeneous viscous liquid minielastin droplets spontaneously matured into a solid structure via the heterogeneous nucleation of internal insoluble clusters. Interestingly, this maturation process was decelerated when the domain length at both termini of minielastin decreased (Fig. 2B). Droplet formation by this shorter minielastin was reversible after it was maintained above the transition temperature for at least 100 min (Fig. 2C). Figure 2D shows the average mean square displacement (MSD) of fluorescent particles vs. lag time within droplets of shorter minielastin with increasing incubation time. The α value represents the diffusive exponent, where α = 1 for a simple viscous fluid. Despite no apparent morphological changes to the shorter minielastin, a decrease in the α value corresponding to the viscoelastic transition was observed with prolonged incubation time (Fig. 2D). However, the viscoelastic transition was slower than that of the original minielastin (Fig. 2E). Although the structural changes at the molecular level remain unknown, this study suggests that proper arrangement of domains can control the kinetics of the transition from reversible to irreversible phase separation in LLPS in an artificial protein inspired by tropoelastin.

Fig. 2
figure 2

The terminal hydrophobic length of minielastin modulates maturation. Differential interference contrast images of minielastin (A) and shortened minielastin (B) as a function of incubation time. The amino acid sequences are as follows: 20′: (VPGVGG)5, 24′: (APGVGV)7, 21–23: EAQAAAAAKAAKYGVGTPAAAAAKAAAKAAQFG. C Temperature-induced reversibility of minielastin droplets after 130 min of incubation. The red arrow indicates the temperature above the coacervation temperature of the construct. The blue arrow indicates the temperature below the coacervation temperature of the construct. Scale bars: 10 μm. D MSD particle trajectory against lag time of minielastin (20′-24′-24′). E Comparison of α values between the minielastin constructs. Reproduced from ref. [21] under Creative Commons CC BY-NC-ND 4.0 License. Copyright 2022, The Authors

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In our approach, we designed “double-hydrophobic” type ELPs that mimic the uneven distribution of glycine-rich and proline-rich hydrophobic domains in tropoelastin [22,23,24,25,26,27,28]. These ELPs self-assemble into nanofibers via the formation of coacervates followed by their connections. The following section reviews the formation mechanisms and applications of these nanofibers.

Self-assembly of double-hydrophobic ELPs

When incubated at 40 °C in aqueous solution, tropoelastin self-assembles into a bundle-like structure of anisotropically assembled filaments approximately 5 nm in diameter [29]. These findings suggest that the tropoelastin sequence has its own molecular mechanism for fiber-like self-assembly. A brief look at the domain structure of tropoelastin revealed that proline-rich sequences are localized to the center of the protein and that glycine-rich sequences are located at the ends of the protein (Fig. 1). We hypothesize that the localization of such domains is responsible for the self-assembly of tropoelastin because block copolymers consisting of two or more distinct segments often exhibit diverse self-assembly behaviors. Thus, a double-hydrophobic ELP, named GPG, was designed and genetically engineered to simplify the heterogeneous domain localization of tropoelastin (Fig. 3A) [22]. GPG has a proline-rich central sequence (VPG[V0.8F0.2]G)25 (F: phenylalanine) flanked by glycine-rich sequences (VGGVG)5. An oligohistidine tag was fused to the C-terminus to facilitate its purification by column chromatography. GPG (20 μM) was dissolved in chilled water and then heated to 45 °C, which is above the transition temperature (approximately 20 °C). Circular dichroism (CD) spectroscopy revealed that GPG adopts a predominantly disordered structure containing some β-turns at 16 °C, whereas it immediately transforms into a structure rich in β-turns at 45 °C (Fig. 3B). Continued incubation at 45 °C resulted in the gradual formation of β-sheet structures over the course of one week. Figure 3C shows the time-dependent changes in the assembled GPG structures observed via atomic force microscopy (AFM). Spherical nanoparticles 130 − 200 nm in diameter were observed soon after the temperature was increased to 45 °C. After 1 d, a significant increase in the particle density and a decrease in the average diameter of the particles were observed. In addition, we observed that fibrils began to appear in another field of view. After Day 2, only nanofibers were observed. The mature fibers formed on Day 7 were 20−70 nm in diameter and more than 10 μm in length with a bead-on-string morphology. The nanofiber dispersion remained transparent without the formation of precipitates even after prolonged aging for several months at 45 °C, indicating the high colloidal stability of the nanofibers.

Fig. 3
figure 3

A Amino acid sequence of GPG. B Temperature-dependent CD spectra of the GPG. C AFM images of the assembled structures of GPG formed at 45 °C. (i) Particles formed just after preparation; (ii) particles and (iii) nanofibers formed on Day 1 at different locations on the mica substrate; (iv) nanofibers formed on Day 2 and (v) Day 7; (vi) high-magnification image of nanofibers with a beaded structure on Day 7. D, E CD spectra and AFM or transmission electron microscopy (TEM) images of the assembled structures formed on Day 7 for the proline-rich monoblock (D), glycine-rich monoblock (E), and diblock (F) polypeptides. G Tentative model for the assembly of GPG into nanofibers. Adapted with permission from ref. [22] Copyright 2013, American Chemical Society

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To verify the importance of the double-hydrophobic molecular design, control experiments were performed using either monoblock or diblock polypeptides. The proline-rich polypeptide (VPG[V0.8F0.2]G)25 alone formed β-turn-rich nanoparticles after heating (Fig. 3D), whereas the glycine-rich polypeptide (VGGVG)5 formed β-sheet-rich short fibrils (<50 nm in length) (Fig. 3E). Interestingly, in these block components, the secondary and aggregate structures quickly reached equilibrium and did not change over time. The diblock counterpart PG, where (VGGVG)5 was only fused at the C-terminus of (VPG[V0.8F0.2]G)25, gradually formed long nanofibers such as GPG, but the β-sheet content in the nanofibers was less than that of GPG (Fig. 3F). In addition, nanofibers were formed via a similar process in a derivative called GPPG, where the proline-rich sequence (VPG[V0.8F0.2]G)25 is repeated twice via a short linker sequence [27]. However, the diameter of the nanofibers determined via AFM was slightly greater, at 55 ± 11 nm for GPPG and 42 ± 8 nm for GPG, suggesting that the molecular size is related to the fiber diameter. Taken together, a tentative model of GPG nanofiber formation is proposed, as shown in Fig. 3G. Upon heating, the GPG phase separated to form coacervates. The GPG then rearranged such that hydrophilic oligohistidine tags induced the adjacent (VGGVG)5 segments to locate near the surface of the coacervate (particle), resulting in compaction of the particle. The (VGGVG)5 segments formed intermolecular β-sheets that interdigitated as the particles assembled into fibers. The relatively slow formation of the β-sheet may be due to the reduced motility of (VGGVG)5 linked to (VPG[V0.8F0.2]G)25.

Nanofiber formation was reversible to some extent. When the GPG nanofiber dispersion (45 °C, 7 d) was cooled to 16 °C for 1 h, isolated particles, short chains of particles, and some remaining nanofibers were observed via AFM. However, complete disappearance of the nanofibers did not occur even after 24 h of incubation at 16 °C.

The self-assembly of GPG is affected by the addition of trifluoroethanol (TFE) [23], an organic solvent that is known to increase the content of α-helices and β-sheets in peptide secondary structures by providing a low dielectric environment that facilitates the formation of hydrogen bonds [30]. The formation of GPG beaded nanofibers was accelerated in a TFE/water mixture up to 30% (v/v) TFE; it was completed as fast as 3 h at 30% TFE even at 16 °C because of the fast formation of β-sheet structures. Nanofiber formation was disrupted, and only irregular aggregates formed at 60% TFE with the formation of α-helix structures.

Functional derivatives of double-hydrophobic ELPs and their applications

Because GPG consists of 196 amino acid residues, the addition of a short peptide at the terminus does not inhibit its ability to form nanofibers [24,25,26]. For example, the KAAK (A: alanine) peptide motif, which is found in the crosslinking domain of tropoelastin, was fused at the C-terminus of GPG [24]. The primary amines in the KAAK motif were chemically crosslinked with bis(sulfosuccinimidyl) suberate (BS3) after nanofiber formation. The crosslinked nanofibers were stable against temperature changes across the transition temperature, whereas their uncrosslinked counterparts partially decomposed at 16 °C.

A derivative with the KAAKGRGDS (R: arginine, D: aspartic acid, and S: serine) peptide at the C-terminus of GPG (GPG-RGD) was constructed [25]. GRGDS is a fibronectin-derived cell-binding peptide that has high affinity for integrins α5β1 and αvβ3. The GPG-RGD nanofibers were coated onto cell culture plates by simply drying the nanofiber dispersion. Murine embryonic NIH/3T3 fibroblasts adhered to and proliferated well on the GPG-RGD nanofiber surface as well as on the positive control fibronectin. In contrast, cell adhesion and proliferation on the nanofibers of GPG or GPG with the KAAK motif were comparable to those on polystyrene cell culture plates. These results indicate that the hydrophilic GRGDS motif was exposed on the fiber surface and effectively interacted with the cells.

Currently available synthetic vascular grafts composed of expanded polytetrafluoroethylene or poly(ethylene terephthalate) represent useful alternatives to diseased vessels. However, they are not suitable for smaller-diameter (<6 mm) applications owing to the loss of patency over time [31, 32]. Elastin, a major ECM protein within the arterial tunica media, has the ideal properties required for synthetic vascular grafts, including antithrombogenicity [33, 34], maintenance of the vascular smooth muscle cell (SMC) phenotype [35, 36], and appropriate mechanical properties [37]. However, the application of biologically derived elastin has been limited by poor processability and substantial batch-to-batch variation. The calcification of elastin after implantation has also been observed in animal models [38]. GPG has a chemical composition similar to that of elastin, can reproduce a fiber-like ECM structure, and may be a candidate material for small-diameter vascular grafts. To further make GPG a suitable material for vascular grafts, we constructed a novel derivative with REDV (E: glutamic acid) peptide motifs [26]. Early endothelialization, like that of biological blood vessels, is another important requirement for vascular graft materials. REDV is a peptide that is specifically recognized by the integrin α4β1 present in endothelial cells (ECs). The biological characteristics of the REDV-modified GPG nanofibers were compared with those of the unmodified GPG, GPG-RGD, collagen and cell culture glass nanofibers. Among these, REDV-modified GPG nanofibers exhibited the preferred biological properties for vascular graft materials: (i) inhibition of platelet adhesion and activation, (ii) EC adhesion and proliferation and (iii) maintenance of SMCs in a contractile phenotype to prevent cell overgrowth, leading to intimal hyperplasia (Fig. 4).

Fig. 4
figure 4

A Platelet adhesion to each surface. The surfaces were allowed to contact the platelet suspensions for 1 h. The platelets were classified into three types according to the number of pseudopodia: Type 1, 0; Type 2, 1–2; and Type 3, more than 2. B Proliferation of human umbilical vein endothelial cells (HUVECs) and C proliferation of human umbilical arterial smooth muscle cells (HUASMCs) on each surface. D Relative α-smooth muscle actin (αSMA) gene expression in HUASMCs on Day 7. αSMA is a contractile protein used as an early marker of the smooth muscle lineage during differentiation. The level of αSMA expression in HUASMCs cultured on glass was set at 1.0. *P < 0.05, **P < 0.01. E Schematics showing the biological properties of the GPG-REDV nanofibers. Reproduced from ref. [26] under Creative Commons CC BY 4.0 License. Copyright 2022, The Authors

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Hydrogels formed via LLPS of tropoelastin and ELPs

Hydrogels composed of tropoelastin and ELPs are attracting great attention as scaffold materials for regenerative medicine because of their cytocompatibility and controllable mechanical properties [39]. LLPS has often been used for the preparation of hydrogels. For example, Weiss et al. chemically crosslinked coacervate droplets of recombinant human tropoelastin (rTE) using BS3 to produce an elastic hydrogel [40]. The attachment and proliferation of epithelial cells were supported in vitro, whereas the degree of cellular penetration within the hydrogels was limited. To overcome these limitations, photocrosslinked hydrogels suitable for in situ cell encapsulation have also been prepared using methacrylated tropoelastin (MeTro) [41]. MeTro hydrogels have been used as sealants for surgical applications [42] and to engineer biomimetic cardiac tissue constructs [43].

Physically crosslinked elastin-like hydrogels were also constructed via LLPS. For example, (VPGXG)90 (X: V, G, A at a ratio of 5: 3: 2) coacervated at 37 °C formed a gel-like phase with a complex modulus |G*| of 80 Pa [44]. Chondrocytes cultured in the gel-like phase maintained their phenotype. Amphiphilic ELP block copolymers with different temperature responses between the blocks were used to obtain a more continuous gel phase [45, 46]. The excellent blood-contacting properties of the block ELP were demonstrated in a primate arteriovenous shunt model [47]. These hydrogels elicited a minimal inflammatory response and displayed robust stability in vivo for more than a year [48].

Extremely stiff physical hydrogels were obtained through a mechanism known as arrested phase separation of concentrated solutions of an ELP [49]. Typically, when solutions of ELPs are heated, they become turbid, and macrophase-separated polypeptide-rich aggregates form. However, by changing the third position of XPGVG from G to A, the phase separation process is arrested at the nanoscale, resulting in the formation of a bicontinuous network in solution. This occurs because the G-to-A substitution impedes the chain dynamics of the folded state in the coacervate [50]. For example, a hydrogel from ([I0.2V0.8]PAVG)120 (I: isoleucine) at 20 wt% has a remarkably high storage modulus G’ of over 1 MPa. A hydrogel was also formed from a telechelic derivative with flanking sequences containing cysteine (C) and RGD cell adhesive sites [51]. Oxidation of the thiol moieties allowed for chain extension of the ELP, leading to the formation of an extensible gel. The chondrocytes encapsulated in the hydrogels remained viable for 28 d.

Physically crosslinked ELP hydrogels are usually obtained at relatively high polypeptide concentrations (>5 wt%). In contrast, double-hydrophobic ELPs that can form nanofibers yield a physical hydrogel at less than 1 wt% [27, 28]. Dynamic rheology measurements revealed that the nanofiber dispersion of GPG with an extremely low concentration (0.034 wt%) exhibited solid-like behavior with a storage modulus G’ greater than the loss modulus G” over a wide range of angular frequencies [27]. This phenomenon is attributed to the formation of high-aspect-ratio (>500) nanofibers, through which percolated networks are formed in solution. When the nanofiber concentration was increased above 0.2 wt%, the hydrogel lost apparent fluidity, and its G’ increased as a function of the concentration (Fig. 5A) [28]. In this case, the GPG hydrogel exhibited similar mechanical properties regardless of the presence or absence of the terminal RGD sequence and could be used to change only the bioactivity while maintaining the same mechanical properties. The hydrogels also exhibit a reversible sol‒gel transition, in which they liquefy upon shaking and then recover their gel state upon standing (Fig. 5B). Figure 5C shows the results of the recovery behavior of G’ and G” by applying cyclic strain to a gel containing 0.5 wt% GPG-RGD. When 100% strain (γ = 100%) was applied to the gel, the relative relationship between G’ and G” was reversed, and the viscous property exceeded the elastic property. When γ = 0.5% was applied, G’ > G” and the elastic properties were restored. This suggests that when a large strain is applied, the physical crosslinking points of the nanofibers dissociate and become viscous. When the strain is removed, the fibers again form crosslinking points and become elastic. This self-healing property can be used to create a three-dimensional culture system in which cells can be embedded and retrieved via mild pipetting. Research on the three-dimensional (3D) culture of cells is currently underway.

Fig. 5
figure 5

A Storage modulus of the GPG and GPG-RGD hydrogels with various polypeptide concentrations (0.050–0.889 wt%) formed after 1 d at 37 °C. Time sweep measurements (1 Hz, 1% strain) were performed for each sample after being transferred to the sample stage of a rheometer, and the G′ values at 3600 s were plotted. The dashed lines represent fits to the data points; the equation of the line for the GPG is G′ = 3.48 × 103C1.53, R2 = 0.984, and for the GPG-RGD: G′ = 2.76 × 103 C1.65, R2 = 0.947). B Photograph of the sol‒gel transition of the GPG hydrogel (0.4 wt%). C Changes in G′ and G″ of the 0.5 wt% GPG-RGD hydrogel formed after 7 d in step-strain sweep dynamic rheological measurements (1 Hz). Reproduced from ref. [28] under Creative Commons CC BY 4.0 License. Copyright 2021, The Authors

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Conclusions and perspective

Tropoelastin and ELPs are representative polymers that exhibit LCST-type LLPS and form coacervates. Since the 1970s, these polymers have been known to form coacervates. However, research into their molecular mechanisms and dynamics has made remarkable progress in recent years. Although the phase transition behavior of ELPs with simple VPGXG repeat sequences is considered reversible, coacervates of synthetic proteins that more closely mimic tropoelastin have been found to undergo irreversible maturation depending on the timescale observed. In particular, double-hydrophobic ELPs that mimic the uneven distribution of the glycine-rich and proline-rich hydrophobic domains of tropoelastin can form anisotropic nanofibers via coacervation followed by molecular reorganization. The biological function of nanofibers is customized by binding functional peptide motifs to double-hydrophobic ELPs, and nanofibers with REDV peptide motifs are particularly promising as components of small-diameter vascular grafts. Furthermore, the nanofiber dispersion has the properties of a physically crosslinked hydrogel, showing a reversible sol‒gel transition in response to the application of shear stress. The viscoelastic properties of the gel make it suitable for a wide range of applications, including 3D cell culture substrates, injectable gels, and bioinks for 3D bioprinting. For future prospects, we are planning the following two specific directions: First, we will fabricate tubular small-diameter vascular grafts and evaluate their mechanical properties in accordance with the ISO7198 standard. If mechanical properties similar to those of autologous blood vessels are obtained, we will move on to preclinical studies using animal models. Then, we will systematically investigate how the mechanical properties and the ratio of functional peptide motifs of the hydrogel affect cell proliferation and differentiation via the use of a double-hydrophobic ELP hydrogel as a 3D cell culture substrate. These fundamental findings could help to obtain guidelines for the design of appropriate hydrogels for future regenerative medicine applications.