Cell‐Like Synthetic Supramolecular Soft Materials Realized in Multicomponent, Non‐/Out‐of‐Equilibrium Dynamic Systems

Abstract Living cells are complex, nonequilibrium supramolecular systems capable of independently and/or cooperatively integrating multiple bio‐supramolecules to execute intricate physiological functions that cannot be accomplished by individual biomolecules. These biological design strategies offer valuable insights for the development of synthetic supramolecular systems with spatially controlled hierarchical structures, which, importantly, exhibit cell‐like responses and functions. The next grand challenge in supramolecular chemistry is to control the organization of multiple types of supramolecules in a single system, thus integrating the functions of these supramolecules in an orthogonal and/or cooperative manner. In this perspective, the recent progress in constructing multicomponent supramolecular soft materials through the hybridization of supramolecules, such as self‐assembled nanofibers/gels and coacervates, with other functional molecules, including polymer gels and enzymes is highlighted. Moreover, results show that these materials exhibit bioinspired responses to stimuli, such as bidirectional rheological responses of supramolecular double‐network hydrogels, temporal stimulus pattern‐dependent responses of synthetic coacervates, and 3D hydrogel patterning in response to reaction–diffusion processes are presented. Autonomous active soft materials with cell‐like responses and spatially controlled structures hold promise for diverse applications, including soft robotics with directional motion, point‐of‐care disease diagnosis, and tissue regeneration.


Introduction
Living cells are multicomponent, nonequilibrium supramolecular systems composed of a myriad of bio-supramolecules, including proteins, (deoxy)ribonucleic acids (DNA/RNA), lipids, and metabolites. [1]iving cells can integrate multiple biosupramolecules independently and cooperatively to execute elaborate physiological functions, including metabolism, motility, immune responses, and neural activity, which cannot be achieved by individual biomolecules.[4] Orthogonal coupling of cytoskeletons with lipid membrane enables cells to rationally execute distinct physiological outcomes, including motility, migration, transportation, and mitosis, without mutual interference.In contrast, crosstalk among individual bio-supramolecules through covalent or noncovalent interactions contributes to the emergence of sophisticated synergistic responses.In intracellular signal transduction systems, such as the mitogen-activated kinase cascade, a series of phosphoproteins arranged along a pathway transmit signals in a sequential, domino-like manner.Kinase cascades effectively amplify signals through reaction and diffusion processes. [1,5]8][9][10] In bacterial cells, the spatial position of cell division is precisely determined by the oscillation of the Min protein complex between cell poles, which is driven by the assembly and disassembly of the protein complex coupled with its diffusion across the bacterial cells. [11]These biological design strategies provide valuable clues for the development of synthetic supramolecular systems with cell-like responses, functions, and spatially controlled hierarchical structures.
[14] To date, biological molecules have inspired the construction of numerous types of synthetic supramolecules, including host-guest chemistry-based artificial enzymes, [15][16][17] metal-organic cages and frameworks with enzyme-mimetic nano-sized spaces, [18][19][20][21][22] vesicles and coacervates for compartmentalization, [23][24][25][26][27][28] and nanofibers and gels resembling cytoskeletons and the extracellular matrix. [13,29]lthough the assembly of individual molecules has been extensively investigated, the interplay and networking among supramolecules remain at the early stage of investigation.[32][33][34][35] These approaches would enable the development of next-generation smart materials that are hierarchically structured with anisotropic arrangements of multiple supramolecules, thus allowing unique nonlinear responses to stimuli and cell-like signal amplification.To realize this systems chemistry approach, supramolecular chemists now face the challenge of developing methods for constructing multiple types of supramolecules in an orthogonal manner, especially in an aqueous environment; regulating interactions among the orthogonal supramolecules; and integrating chemical and enzymatic reactions to prevent the interference of reactive functional groups.In this perspective, we describe our recent progress in constructing multicomponent supramolecular soft materials through the hybridization of supramolecules, such as self-assembled nanofibers, gels, and coacervates, with other functional molecules, including polymer gels and enzymes (Figure 1).Moreover, we present results showing that these materials exhibit bioinspired responses to stimuli, such as bidirectional rheological responses of supramolecular double-network hydrogels, temporal stimulus pattern-dependent responses of synthetic coacervates, and 3D hydrogel patterning in response to reaction-diffusion processes.8][39][40][41][42]

General Design of Building Blocks and Self-Assembly Strategies for Multicomponent and Non-/Out-of-Equilibrium Dynamic Systems
In this section, we highlight the structural design of building blocks for supramolecular hydrogels and coacervates described in this Perspective (Figure 2 top).Stimuli-responsive peptide-type hydrogelators of di-or tri-phenylalanine which have been extensively used as a self-assembling moiety in aqueous solutions [43][44][45][46][47][48] are one of the key players.The peptide-type hydrogelators form into crystalline nanofibers with a -sheet like structure, mainly driven by - interactions between benzene rings and hydrogen bonds between amide groups.We previously discovered the addition and deletion of the hydrophobic moiety at the Nterminus of these peptides through chemical reactions can induce the sol-to-gel and gel-to-sol transitions, respectively. [33,49,50]ipid-type hydrogelators, another key player, are amphiphiles that have a hydrophilic head group (e.g., phosphate and Nacetylgalactosamine, GalNAc) and two cycloalkyl groups as hydrophobic tails. [51,52]Due to the too-short hydrophobic tails, the lipid-type hydrogelators can't form spherical vesicles, but instead, form 1D nanofibers mainly through hydrophobic interactions between the cycloalkyl chains and hydrogen bonds between fumaramide groups.The interaction modes are remarkably different from those of peptide-type hydrogelators.Also, we recently developed a new structural motif, tert-butyl diphenylalaninate (FF-OtBu) to generate liquid-liquid phase separation (LLPS). [53,54]A bulky tBu group newly introduced at the Cterminus of a diphenylalanine peptide may suppress -sheetbased nanofiber formation, and induce LLPS.Unlike the peptidetype hydrogelators, several types of hydrophilic functional groups are attached at the N-terminus of the FF-OtBu motif and we have found unique coacervates composed of a short peptide.Furthermore, we integrated other functional molecules, including enzymes (reaction catalysts), synthetic covalent polymers (stiff structure motif), porous materials (molecular recognition), and metal ions (coordination bonding), into supramolecular hydrogels and coacervates, by which sophisticated stimulus-responsive systems are fabricated.
To realize cell-like responses, functions, and hierarchical structures using these building blocks, we employed the four selfassembly strategies (Figure 2 middle, bottom).The first involves the orthogonal self-assembly of multiple supramolecules through self-sorting phenomena.We demonstrated that peptideand lipid-type hydrogelators self-assemble orthogonally without mixing each other, resulting in the formation of self-sorting double-network (SDN) hydrogels.The self-sorting phenomena allow the rational integration of stimulus responses from these peptide-and lipid-type nanofibers without mutual interference (Section 3.1).Four control factors were identified to date for selfsorting: i) distinct chemical structures and driving forces of selfassembly, ii) the same charge (in our case, anionic peptide-and lipid-type hydrogelators were used), iii) the moderate hydrophobicity of the peptide-type hydrogelators, iv) distinct formation kinetics (lipid-type hydrogelators self-assemble first).The second strategy relies on post-assembly fabrication (PAF) to extend the range of stimulus responses in SDN hydrogels by the addition of other functional molecules (Section 3.2).To retain the self-sorting network, the functional molecules are implemented after the construction of SDN hydrogels.The PAF strategy is also applicable to in situ degradation and formation of peptide-type nanofibers in the presence of lipid-type nanofibers, which enables control of the self-sorting network (Section 4.2).The third strategy is based on a reaction-diffusion system, which is a widely-used method to spontaneously generate spatiotemporal patterns.In this Perspective, we describe the emergence of propagating waves of supramolecular nanofibers, whose generation and degradation are controlled under concentration gradients of distinct chemical stimuli (Section 4.1).The interplay between radical polymerization and its inhibition reaction results in temporal stimulus pattern-dependent responses in synthetic coacervates (Section 3.4).The last employs microscopic/macroscopic phase separation, which is a potent approach for the development of hierarchically structured soft materials.In particular, phase separation enables the construction of 3D patterned hierarchical hydrogel materials through the combination of top-down fabrication methods (Section 4.4).

Emergence of Unique Cell-Like Responses by Incorporating Multiple Supramolecules
This section describes synthetic systems exhibiting unique responses to stimuli, which are obtained by integrating multiple supramolecules and/or other functional molecules in an orthogonal and/or cooperative manner.[57] Subsequently, they developed a platform by incorporating thermosensitive liposomes encapsulating enzymes to release the small organic molecule covalently attached to the enzyme-responsive hydrogelator of this orthogonal self-assembly system. [58]Controlled heating of this three-component system liberates the enzyme into the gel matrix, and subsequent hydrolysis of the hydrogelator releases the small molecule with a release rate controlled by the heating time.We constructed a hydrogel system capable of fluorescence sensing of biologically relevant polyanions and polycations by integrating porous inorganic materials (montmorillonite and mesoporous silica particles (MCM41), respectively) with lipidtype supramolecular fibers, which allows visualization of analyteinduced exchange and location shift of fluorescent dyes in this multicomponent hydrogel system. [59,60]These pioneering examples indicate that integrating multiple supramolecules can be applied to diagnostic disease-sensing devices and controlled drugrelease systems.

Bidirectional Rheological Responses of Self-Sorting Supramolecular Nanofibers
Cytoskeletons, such as actin filaments and microtubules, exhibit self-sorting, which can be applied as a promising approach to rationally integrate supramolecules into a single synthetic system. [51, To deonstrate this approach, we constructed SDN hydrogels with bidirectionally tunable rheological properties (Figure 3a). [77]Previously, we found that peptideand lipid-type hydrogelators are promising pairs to achieve selfsorting in an aqueous environment because of their distinct chemical structures and molecular interactions. [73,75]To prepare SDN hydrogels, NPmoc-F(F)F and Phos-cycC 6 were employed as peptide-and lipid-type hydrogelators, respectively (Figure 3b,c).NPmoc-F(F)F nanofibers decompose in the presence of a reductant (sodium dithionite) through nitrobenzyl group reduction followed by 1,6-elimination to produce a more hydrophilic F(F)F dipeptide (Figure 3b).In contrast, Phos-cycC 6 converts into a more hydrophobic HO-cycC 6 in response to the alkaline phosphatase (AP) enzyme to become a stiffer hydrogel (Figure 3c).Finally, these two nanofibers integrate through selfsorting (Figure 3d).Rheological analysis showed that the resultant self-sorting hydrogel exhibits bidirectional responses.Specifically, the storage modulus decreased from 187 to 2 Pa after treatment with the reductant but increased to 1450 Pa after the addition of AP (Figure 3e).High-performance liquid chromatography (HPLC) analysis confirmed that the reductant and AP selectively react with the corresponding hydrogelators.In addition, we succeeded in controlling the release rate of proteins embedded in the SDN hydrogels (Figure 3f).For example, the IgG antibody was slowly released from nontreated self-sorting hydrogels with a release rate of 40 ± 12% over 10 h, which was bidirectionally changed to 87 ± 19% and 14 ± 2% after treatment with the re-ductant and AP, respectively.It is clear that self-sorting can be applied to the design of smart soft materials that are responsive to multiple stimuli for controlled drug release and cell culturing.

Extension of Stimulus Responses of Self-Sorting Supramolecular Hydrogels by Post-Assembly Fabrication
Although constructing SDN hydrogels by self-sorting is a powerful approach, it requires delicately balancing the noncovalent interactions of hydrogelators.Therefore, there is currently a limited variety of SDN hydrogels.To extend SDN hydrogels, we proposed a PAF strategy, in which functional molecules and/or enzymes are added to the constructed SDN hydrogel (Figure 4a). [78]Sarcosine oxidase (SOx) enzyme and Ca 2+ ions were selected for the PAF of the BPmoc-F 3 /Phos-cycC 6 SDN hydrogel.SOx generates H 2 O 2 , glycine, and formaldehyde from sarcosine, and generated H 2 O 2 decomposes BPmoc-F 3 nanofibers (Figure 4c).In contrast, Ca 2+ ions interact with the phosphate groups on the surface of Phos-cycC 6 nanofibers to induce a sol-to-gel transition through multivalent coordination bonding (Phos-cycC 6 forms nanofibers in the solution state).Ca 2+ -bound Phos-cycC 6 nanofibers respond to ATP because Ca 2+ ions have a stronger affinity for the triphosphate group of ATP than Phos-cycC 6 , leading to a gel-to-sol transition.Overall, the multicomponent SDN hydrogel prepared with PAF using SOx and Ca 2+ ions exhibits an ANDtype gel-to-sol response by the action of biologically relevant molecules, sarcosine and ATP.Confocal imaging [85,86] showed that the self-sorting network is retained even after the PAF process (Figure 4b).The resultant multicomponent SDN hydrogel undergoes a macroscopic gel-to-sol transition in response to both sarcosine and ATP, while the hydrogel maintains its gel state following the action of either sarcosine or ATP (Figure 4d).The multicomponent SDN hydrogel is a suitable AND-type active matrix for the controlled release of fluorescently modified IgG antibodies (Figure 4e-g).Notably, the one-step mixing protocol, in which Ca 2+ ions are mixed with a mixture of two hydrogelators before SDN formation, produces a white suspension instead of the SDN hydrogel, indicating that PAF is an essential step in the construction of multicomponent SDN hydrogels with extended stimulus responses.

Nonenzymatic Protein-Dependent Responses of Multicomponent Supramolecular-Polymer Composite Hydrogels
In addition to enzymes, nonenzymatic proteins, such as antibodies, often serve as biomarkers for several diseases.[93][94] We successfully prepared a supramolecular-polymer composite hydrogel that can respond to nonenzymatic proteins by integrating an enzyme-responsive supramolecular hydrogelator with an enzyme-activation system. [95]In particular, we designed an enzyme-responsive hydrogelator, APmoc-F(CF 3 )F, with an acetoxybenzyl group at the N-terminus (Figure 5a).APmoc-F(CF 3 )F undergoes a gel-to-sol transition in response to bovine carbonic anhydrase II (bCAII).To elicit a response to nonenzymatic proteins, we designed an enzyme-activation trigger (EAT) molecule consisting of a bCAII inhibitor (benzenesulfonamide) and a ligand of the target nonenzymatic protein connected by a short linker (Figure 5c). [96]bCAII activity is tentatively inhibited in the presence of EAT and recovered after the addition of the target protein owing to the competition between bCAII and the protein for binding to EAT.Re-activated bCAII then de-composes APmoc-F(CF 3 )F nanofibers.Overall, integrating the EAT system enables APmoc-F(CF 3 )F nanofibers/hydrogels to respond to nonenzymatic proteins (Figure 5d).
Covalent polymer gels (agarose) can be hybridized with supramolecular hydrogels and an EAT molecule to construct soft materials that release proteins in response to nonenzymatic proteins (Figure 5b).Confocal images of the orthogonal network of the APmoc-F(CF 3 )F/agarose composite hydrogel indicated that stimulus-responsive APmoc-F(CF 3 )F is retained in the composite hydrogel (Figure 5e).Indeed, HPLC analysis showed that 45% of APmoc-F(CF 3 )F in the composite hydrogel, which contains bCAII and an EAT molecule (biotin as the ligand for avidin), decomposes in the presence of avidin, a model nonenzymatic protein, while negligible decomposition occurs in the absence of avidin and under a competitive environment for biotin.Notably, the composite hydrogel maintains its gel state even after APmoc-F(CF 3 )F decomposes, suggesting that the agarose network orthogonally works to retain the structural stability of the composite hydrogel.To realize the controlled release of proteins embedded in the composite hydrogels, we subsequently evaluated the release of entrapped myoglobin (Mb), serving as the model protein.Composite hydrogels containing Mb were immersed in a buffer solution for 3 h, and SDS-PAGE analysis of the supernatant indicated that 97% of Mb was entrapped in the composite hydrogel.In contrast, only 6.6% of Mb was retained in the single-component agarose gel, indicating that Mb was entrapped by APmoc-F(CF 3 )F nanofibers.We then quantified the avidin-responsive release of embedded Mb from the multicomponent composite hydrogel (Mb/bCAII/EAT⊂APmoc-F(CF 3 )F/agarose), revealing that 75% of Mb was released in the presence of avidin, whereas only 2.3% of Mb was released in the absence of avidin (Figure 5f,g).We found that this system can be extended to a different combination by designing a corresponding EAT molecule: anti-DNP antibody as the target protein and RNase A (a promising protein drug for cancer) as the embedded protein (Figure 5h).Our composite system (bCAII/EAT⊂APmoc-F(CF 3 )F/agarose) can function as an antibody-responsive RNase A release matrix, which can potentially be used for the release of protein-based pharmaceuticals controlled by a distinct biomarker protein (antibody).Overall, integrating distinct multiple chemical systems in concert is a promising approach to producing autonomous intelligent soft materials for drug delivery, cell culturing, and regenerative medicine.

Temporal Stimulus Pattern-Dependent Responses of Synthetic Coacervates
Living systems can decode static signals as well as temporally dynamic signals that vary in frequency and duration into different phenotypic responses. [5,97]For example, neural progenitor cells maintain their multipotent state under the oscillatory expression of three different types of transcription factors, while the cells differentiate into three distinct cell types (neurons, astrocytes, and microglia) when oscillatory expression changes to a sustained expression pattern. [98]To date, a wide variety of synthetic supramolecules that are responsive to static stimuli have been reported, whereas synthetic systems that can decode the temporal patterns of dynamic signals are rare.In 2022, we reported the differentiation of a synthetic dipeptide-based coacervate in response to the temporal pattern of a stimulus (Figure 6a). [53]The key to achieving this outcome is the construction of a coacervate using a synthetic dipeptide comprising a self-assembling FF-OtBu core and a cationic phenylpyridinium group at the N-terminus (PhePy-FF-OtBu) to accommodate both light-triggered radical polymerization and its competitive reaction (Figure 6b).PhePy-FF-OtBu coacervate droplets can absorb anionic molecules through electrostatic interactions.The coacervate serves as a unique reaction environment for the light-triggered radical polymerization of an anionic methacrylate monomer (sulfoMA) in the presence of an anionic initiator (LAP) (Figure 6c).The coacervate environment and temporal light pattern affect the efficiency of radical polymerization, namely, monomer consumption and the degree of polymerization (DP n ), which is conducted in a competitive environment with molecular oxygen.Additionally, the produced anionic polymers interact with the cationic coacervate droplets through multivalent electrostatic interactions to modify the coacervate property.Radical polymerization was triggered by irradiating the coacervate of PhePy-FF-OtBu, including sulfoMA and LAP, with dual-frequency pulsed light (9 and 0.5 Hz) in the air. 1 H nuclear magnetic resonance (NMR) and size-exclusion chromatography measurements confirmed that radical polymerization occurs under 9 Hz pulsed-light irradiation (monomer conversion: 22 ± 2%, DP n : 63 ± 5), while negligible polymerization proceeds under 0.5 Hz pulsed-light irradiation.As a control experiment, radical polymerization was triggered with 0.5 Hz pulsedlight irradiation under Ar (monomer conversion: 47 ± 9%, DP n : 120 ± 30), suggesting that competition between sulfoMA monomers and O 2 for the reactive radical species is crucial for temporal pattern-dependent polymerization.Remarkably, coacervate droplets transform into multiphase droplets (coacervate-incoacervate droplets) under 9 Hz pulsed-light irradiation, while  negligible changes occur under 0.5 Hz pulsed-light irradiation (Figure 6d).Fluorescence recovery after photobleaching (FRAP) analysis revealed that both the mobile fraction and half recovery time after 9 Hz irradiation are significantly different from those of the nonirradiated and 0.5 Hz irradiated samples.Moreover, the values suggested that the inner environment of the coacervate droplets becomes gel-like after 9 Hz irradiation (Figure 6e).Collectively, the results indicated that the morphology and physical properties of dipeptide-based coacervates can differentiate in response to temporally distinct pulsed-light patterns.[101] A plausible mechanism of this ultrasensitive response is as follows: The threshold amount of reactive radical species produced at the lower frequency (0.5 Hz) is quenched by molecular oxygen diffused from the air, whereas the large amount of radicals produced at the higher frequency (9 Hz) is not completely quenched by molecular oxygen, which allows radical polymerization to proceed (Figure 6f).Overall, cell-like responses to temporal stimulus patterns emerge by combining the polymer formation reaction and the polymer inhibition reaction involving molecular oxygen diffusion into the viscous coacervate layer.
][104][105][106][107][108][109][110] We recently found that a coacervate comprising PEG 9 -FF-OtBu with a thermally responsive nonaethylene glycol group at the N-terminus of FF-OtBu undergoes a pathway-dependent phase transition (Figure 7a,b). [54]In the low temperature (LT) phase, PEG 9 -FF-OtBu forms coacervate droplets with a 3D sponge-like inner network, whereas in the high temperature (HT) phase, the coacervate droplets transform into a homogenous structure through a coacervate-to-coacervate transition (Figure 7c,d).Time-lapse confocal imaging revealed that the LT-to-HT or HT-to-LT phase transition produces distinct intermediate structures.In the path from LT to HT, thin-layer membrane structures bud at the periphery of the coacervate, and vacuoles (possibly filled with buffer solution) form inside the coacervate (Figure 7e).Subsequently, the vacuoles move to the edge of coacervate droplets, and concurrently the inner structure changes from a sponge-like network to a homogeneous structure.These changes are completed within 5 min.After incubation, the membranes suddenly burst to afford spherical coacervate droplets.In contrast, along the HT-to-LT pathway, the inner coacervate structure gradually transforms into a spongelike network over 60 min (Figure 7f).These phase transitions can be reasonably explained by water release and uptake of the PEG 9 moiety in response to the temperature change.The HT-to-LT phase transition requires more time than the LT-to-HT phase transition to reach a thermally equilibrated state, probably because of the slow water/buffer uptake into the coacervate droplets for hydration of the PEG 9 chain.

Spatiotemporal Control of Static/Dynamic Supramolecular Structures
Living organisms are constructed with 3D hierarchical structures spanning a wide range of scales, from biomolecules to organelles, cells, and organs.Importantly, these structures are tightly linked with sophisticated biological functions. [1]These hierarchical structures are prepared by dynamically controlling the formation and degradation of biomolecules and their supramolecular assemblies, which are dependent on the interplay among diverse (enzymatic) reactions, protein-protein interactions, and self-assembly processes in reaction-diffusion systems. [6,7,9]onstructed to mimic naturally occurring systems, spatially patterned synthetic supramolecular systems in 2D and 3D undoubtedly have the potential to serve as functional materials for biomedical applications, such as controlled drug release, tissue engineering, and soft robotics.To date, top-down approaches, including molding, photo-patterning, and soft 3D printing, have been widely used to fabricate spatially patterned supramolecular soft materials. [36,93]Adams and coworkers used photo-triggered spatial patterning to construct SDN hydrogels comprising lightresponsive and nonresponsive hydrogelators. [68]Owing to selfsorting, localized irradiation of the hydrogel through a photo mask selectively decomposes light-responsive nanofibers to generate a patterned network.van Esch and colleagues realized hierarchical compartmentalized multicomponent supramolecular hydrogels by self-sorting at both the monomer and fiber levels.Hydrazone formation in a system comprising watersoluble hydrazide and neutral and anionic aldehyde derivatives is accompanied by the in situ generation of multicomponent hydrogelators that self-sort into nanofibers, which further self-sort into separated microdomains at the several tens of micrometers scale, resulting in hierarchically compartmentalized supramolecular hydrogels. [79]][113][114][115][116][117][118][119][120][121] For example, van Esch and coworkers reported the spatially controlled formation of supramolecular hydrogels through a combination of reaction-diffusion, and self-assembly of hydrogelators in a polymer hydrogel matrix. [113]In this approach, nonassembling precursors (aldehyde and hydrazine derivatives) diffuse from opposite sides of the inert agar or Ca 2+ -alginate gel matrix and react at the crossing front to produce tris-hydrazone hydrogelators, which subsequently self-assemble into supramolecular hydrogels.This reaction-diffusion method enables the fabrication of macroscopic hydrogel objects with sizes varying from several hundred micrometers to centimeters.Prins and colleagues demonstrated that ATP-templated self-assembled vesicles locally accumulate near the injection point of ATP in an agarose hydrogel matrix. [122]In contrast to the conventional reactiondiffusion method (diffusion-then-assembly protocol), local self-assembly of large vesicles with small diffusion coefficients depletes small-molecule amphiphiles, resulting in the diffusion of amphiphiles toward the injection point along a concentration gradient (assembly-then-diffusion protocol).This method allows for progressive local accumulation of vesicles through repetitive ATP injection.Compared with homogeneous gel matrices, the resultant inhomogeneous gel matrix upregulates chemical reactions catalyzed by localized ATP-templated assemblies.In this section, we highlight the recent results of our studies on the spatial control of multicomponent supramolecular systems through nonequilibrium processes over a wide range of scales, from nanofibers to bulk hydrogels.

Propagating Wave of Supramolecular Nanofibers Undergoing Spatiotemporally Coupled Formation and Degradation
There is a rich variety of dynamic (transient) patterns in living cells.A representative is the propagating wave of actin filaments at lamellipodia, in which polymerization and depolymerization are spatiotemporally controlled by reactions (including ATP hydrolysis), protein-protein interactions, and the diffusion of actin monomers and interacting proteins. [2,123,124]We succeeded in generating a propagating wave of synthetic supramolecular nanofibers undergoing spatiotemporally coupled growth and shrinkage (Figure 8a). [115]on-interfering chemical stimuli were employed to induce nanofibers to undergo stimuli-induced formation and degradation, thus producing the supramolecular propagating wave (Figure 8b).Zn 2+ ions were incorporated into BPmoc-F 3 through coordination bonding with the carboxylate group of BPmoc-F 3 to enhance intermolecular interactions and reduce charge repulsion, thus promoting the formation of BPmoc-F 3 nanofibers.As the degradation stimulus, we selected H 2 O 2 to react with the phenylboronic acid group to convert BPmoc-F 3 into a more hydrophilic triphenylalanine peptide, resulting in nanofiber decomposition.H 2 O 2 is produced in situ through an enzymatic reaction of glucose oxidase (GOx) and glucose.It is known that Zn 2+ ions do not affect the activity of GOx, and they do not react with H 2 O 2 .Propagating waves emerge along the concentration gradient of the chemical stimuli (Zn 2+ ions and glucose) created by injecting these stimuli to one side of a mixture of BPmoc-F 3 , GOx, and a fluorescent probe.Confocal microscopy was used to visualize the small puncta formed after stimulus addition, the nanofibers growing from one side to the other side of the field of view, and the nanofibers gradually disappearing along the same direction (Figure 8c).The time profile of the fluorescent intensity at distinct x coordinates demonstrated that the time of the maximum fluorescent intensity was delayed as the x-coordinate increased, indicating the formation of a propagating wave of supramolecular nanofibers along the concentration gradient of the stimuli (Figure 8d).The average traveling distance and velocity of the wave were estimated to be 340 ± 40 and 54 ± 8 μm min −1 , and we found that the pushing force was generated by the supramolecular propagating wave, which was determined to be 0.005 pN, from the displacement of microbeads.Moreover, the numerical analysis and simulation based on a reaction-diffusion mechanism provided important insights into the propagating wave: i) The wave velocity is proportional to the formation kinetics of the nanofibers and the diffusion coefficient of the degradation stimulus, ii) the concentration gradient of the degradation stimulus is one of the key parameters for maintaining the propagating wave, and iii) the diffusion coefficient of the supramolecular fibers is small, which is another important factor for generating the propagating wave.

Construction of Interpenetrated and Parallel Self-Sorting Networks
8][129][130][131][132][133][134] We recently demonstrated that controlling fiber formation and degradation (polymerization/depolymerization) can provide supramolecular hydrogels with two distinct SDN network patterns (interpenetrated and parallel). [51]In particular, the seed formation kinetics is a critical factor for the discriminative preparation of the two patterns.
We controlled the formation of peptide-type nanofibers in situ by using dynamic covalent oxime bonding.The reaction of O-benzylhydroxylamine and a peptide-type hydrogelator with a benzaldehyde group at the N-terminus (Ald-F(F)F) afforded a hydrophobic benzyloxime-type hydrogelator (BnOx-F(F)F) to induce a sol-to-gel transition (Figure 9a).Confocal imaging was used to visualize the formation of peptide-type nanofibers following the addition of O-benzylhydroxylamine to the hydrogel composed of Phos-MecycC 5 fibers and Ald-F(F)F monomers (Figure 9b-d).The line plot analysis confirmed that the formed peptide-type nanofibers did not overlap with the lipidtype nanofiber (Figure 9e).The Pearson's correlation coefficient indicated that a negligible correlation existed between these two types of nanofibers, suggesting the formation of an interpenetrated self-sorting network.Time-lapse confocal images showed that the peptide-type nanofibers formed at two distinct nucleation sites: on the surface of lipid-type nanofibers and at the water layer (Figure 9f).At the initial stage (up to 3 min), short peptide-type aggregates/fibers selectively nucleated on the surface of the lipidtype nanofibers.Subsequently, peptide-type nanofibers appeared on the lipid-fiber surfaces and within the water layer.These images suggested that nucleation on the lipid-fiber surface was kinetically favored over nucleation in the water layer.
Confocal microscopic observations also indicated that a decrease in the formation kinetics of BnOx-F(F)F leads to preferential nucleation on lipid-type nanofibers, which provides the parallel self-sorting network.To decelerate the formation kinetics, we employed an oxime-exchange reaction to transform hydrophilic carboxylate oxime-type hydrogelator (CaOx-F(F)F) to BnOx-F(F)F (Figure 9g).Carboxylic acid hydroxylamine was added to the interpenetrated self-sorting hydrogel comprising Ald-F(F)F and Phos-MecycC 5 nanofibers, which decomposed the Ald-F(F)F nanofibers (Figure 9h, left, middle).To this single lipid-network hydrogel, O-benzylhydroxylamine was subsequently added.As expected, peptide-type nanofibers were slowly reformed, and these peptide-type nanofibers were closely aligned but not completely colocalized with the lipid-type nanofibers (Figure 9h, right).The line plot analysis showed that the peak tops of the peptide-and lipid-type nanofibers were slightly out of alignment, although the peak patterns were similar, suggesting that the parallel self-sorting network formed.Time-lapse imaging revealed that 98.6% of the peptide-type nuclei formed on the surface of the lipid-type nanofibers (Figure 9i).Moreover, time-lapse confocal movies revealed that two critical parameters of the formation kinetics of the BnOx-F(F)F nanofibers differed between the oxime-formation and oxime-exchange protocols: the appearance time of short peptide fibers (1.5 min and 1 h 40 min) and the averaged elongation rates (3 ± 3 and 0.06 ± 0.03 μm min −1 ), respectively.

Identification of Four Types of Network Patterns in Supramolecular-Polymer Composite Hydrogels
Differences in the kinetics of self-assembly may enable the production of multicomponent hydrogels with different network patterns.Recently, we discovered that supramolecular-polymer composite hydrogels can adopt four types of network patterns, which are governed by both the order of network formation and interfiber interactions. [119]e used confocal microscopy to investigate the network structures of composite hydrogels composed of agarose and various supramolecular hydrogelators (Figure 10a).The orthogonal network comprising the supramolecular and agarose networks is morphologically similar to those of the single components, i.e., the fibrous network of the supramolecule and the sea-island network for agarose.Additionally, three other networks, classified as interactive networks, are characterized by deviations of either the supramolecular or agarose network from the original morphology of the single component.In the case of the GalNAc-cycC 6 /agarose composite hydrogel, the agarose morphology was altered from the sea-island network to the fibrous network, while the GalNAc-cycC 6 morphology retained the fibrous network.The fibrous agarose network was colocalized with the GalNAc-cycC 6 network (termed interactive type I network).In the Phos-cycC 6 /agarose network, the agarose morphology retained the seaisland network, albeit with smaller islands and void spaces, while the Phos-cycC 6 morphology retained the fibrous network (termed interactive type II network).The DBS-COOH/agarose composite hydrogel, prepared by decreasing the pH, also adopted the interactive type III network.In this hydrogel, the supramolecular network changed into a heterogeneously distributed domain structure (in the absence of agarose, DBS-COOH formed the fibrous network, similar to the other supramolecular hydrogelators), while the agarose morphology retained the sea-island net-work.The central region of the supramolecular domains overlapped with the island region of the agarose network, indicating that the agarose network served as the nucleation site of DBS-COOH.
Time-lapse imaging of the formation processes unveiled two factors governing the generation of the network patterns: the order of network formation and interfiber interactions (Figure 10b).When the agarose network forms first, the self-assembly of LMW hydrogelators is affected by the agarose network.If there are negligible interactions between the supramolecule and agarose, the supramolecule forms an independent network without interference from agarose, resulting in the orthogonal network.If LMW hydrogelators and agarose interact with each other, the interactive type III network forms because the nucleation and growth of supramolecular nanofibers preferentially occur in the island domain of the agarose network.When supramolecular fiber formation is faster than agarose network formation, the supramolecular network impacts the diffusion and network of agarose.If the interaction between the supramolecule and agarose is minimal, polymer diffusion is physically inhibited by the supramolecular network to afford the interactive type II network.In the case of substantial interaction, the interactive type I network is formed through preferential aggregation of the agarose network on the supramolecular nanofibers serving as a scaffold.Therefore, delicate control of the formation kinetics and interfiber interactions is essential to obtain μm-sized network patterns of multicomponent synthetic hydrogels.

Fracture-Induced 3D Patterning of Supramolecular-Polymer Composite Hydrogels
[137][138][139][140] For example, we created fractures to induce network remodeling of supramolecular-polymer composite hydrogels in 3D (Figure 11a). [119]y chance, we discovered that a composite hydrogel spontaneously alters its network on the several-hundred-micrometer scale during incubation.As-prepared NPmoc-F(F)F/agarose hydrogel forms a homogeneously distributed network, and the supramolecular (NPmoc-F(F)F) network gradually transforms into heterogeneously segregated domains with diameters of approximately 500 μm after room-temperature incubation (Figure 11b,c).Concurrently, the agarose network also becomes heterogeneously distributed, impinging the supramolecular network domains.These observations suggested that autonomous network remodeling is similar to phase separation in an aqueous two-phase system.Circular dichroism (CD) spectra indicated that the packing structure of NPmoc-F(F)F nanofibers is another factor of network remodeling.Subsequently, we punctured the hydrogel exhibiting autonomous remodeling with 3D-printed microneedle stamps to induce 3D patterning.For example, using a concentric-patterned microneedle stamp with needle lengths varying from 200 to 1400 μm, we induced macroscopic patterning in the condensed region of NPmoc-F(F)F nanofibers to controlled depths (Figure 11d,e).Fracture-induced network  remodeling using micrometer-precision top-down techniques may facilitate the spatially programmable patterning of functional multicomponent hydrogels in 2D and 3D.
[159] The E-isomer of the benzoylhydrazone group isomerizes into the Z-isomer upon UV-light irradiation and thermally reverts to the E-isomer during incubation at room temperature.The Z-isomer disturbs the packing mode of the peptide nanofibers, and thus the nanofibers destabilize upon light irradiation and thermally reform.The photo-responsive SDN hydrogel was prepared using the PAF protocol.Specifically, benzhydrazide was added to the SDN hydrogel comprising Ald-F(F)F(F) and Phos-cycC 6 to attach the benzoylhydrazone group to the peptide nanofibers.Time-lapse confocal microscopic images were obtained with low-magnification  objectives before and after localized light irradiation ( max : 325 nm) through a line-shaped photomask (line width, 20 μm; interval, 180 μm) (Figure 12c).Photo-irradiation generates dark lines in the peptide-fiber channel but not in the lipid-fiber channel, indicating that the peptide nanofibers are selectively decomposed.Line plot analysis showed that the fluorescence of peptide nanofibers in the irradiated area gradually strengthens during the thermal incubation, and the fluorescence of peptide nanofibers in the nonirradiated area concurrently weakens (Figure 12d).High-magnification confocal laser scanning microscopy images showed that the numerous BHz-F(F)F(F) nanofibers in the photo-irradiated area are regenerated close to peptide aggregates used as seeds.Therefore, the photo-triggered out-of-equilibrium pattern arises from the condensation of peptide nanofibers regenerated in the photo-irradiated areas where peptide monomers are supplied from the nonirradiated area by diffusion (Figure 12e).CD spectroscopic analysis revealed that the as-prepared BHz-F(F)F(F) nanofibers are in the metastable state and the nanofibers reformed after photo-irradiation are in the thermodynamically stable state.
We applied this out-of-equilibrium patterning to create unique photo-triggered patterns, which are inaccessible by conventional photofabrication of materials.For example, a double-line pattern was obtained upon consecutive light irradiation through two dis-tinct alignments of a one-line photomask (line width: 40 μm) and a unique grid-like pattern.Additionally, spatial patterning of non-photo-responsive proteins is possible if the proteins interact with the BHz-F(F)F(F) nanofibers.These results highlight the power of integrating photochemical response, kinetic trapping, and diffusion-controlled self-assembly in multicomponent supramolecular materials, which can facilitate the rational design of synthetic materials with spatiotemporally controlled hierarchical structures and functions.

Conclusion and Future Perspective
In this perspective, we briefly describe our recent achievements in fabricating multicomponent supramolecular systems exhibiting cell-like responses and hierarchical structures by integrating multiple supramolecules and functional molecules in an orthogonal and/or cooperative manner.[162][163][164][165][166][167][168][169][170][171][172][173][174][175] For example, van Esch and coworkers demonstrated the dissipative self-assembly of a supramolecular hydrogel driven by consuming a chemical fuel. [162,176]Methylation of the carboxylate of a hydrogelator precursor by dimethyl sulfate (as the chemical fuel) coupled with ester hydrolysis led to transient self-assembly of the active methylated hydrogelator.According to time-lapse confocal images, at the microscopic level, some fibers stochastically collapsed from their tips, while other fibers both grew and shrank simultaneously over a certain period, which is similar to the dynamic instability of microtubules.Hermans and colleagues induced oscillation of a supramolecular polymer composed of a perylene diimide derivative. [165]This oscillator was driven by a combination of positive feedback (nucleation-elongation-fragmentation) and negative feedback (size-dependent depolymerization) under the continuous addition of a reductant as the chemical fuel.Fletcher and coworkers constructed a chemically fueled micelle oscillator using thiol-disulfide chemistry coupled with the autocatalytic formation and degradation of the active amphiphile in a well-stirred biphasic setup. [173]Otto and coworkers developed a light-driven protometabolic and eco-evolutional system of synthetic self-replicating molecules that can recruit and activate photocatalysts. [169,175]These systems rely on the positive feedback loop involving macrocyclic precursors produced by the photocatalytic oxidation of constituents.Exploiting the interplay among multiple functional supramolecules is a promising strategy for the development of next-generation soft supramolecular materials that are responsive to various stimuli.However, such a systems chemistry approach is in the early stage of development.
Figure 13 shows a potential roadmap toward cell-like soft materials, including the development of synthetic cells, active matter assemblies, and intelligent biomedical materials.It is worth noting that in certain areas, the molecular design guidelines and selfassembly strategies still remain limited: i) expanding multicomponent orthogonal self-assembly pairs and uncovering control factors; ii) exploring multiple synthetic pairs of high-energy substrates and catalysts with high reactivity and substrate specificity; iii) designing elaborate reaction networks for nonlinear reaction kinetics; iv) controlling the balance between the reaction kinetics and diffusion rates; and v) understanding and rationally designing non-/out-of-equilibrium phenomena by numerical simulation and machine learning.[189][190][191][192][193][194][195][196] Because their physicochemical properties and functions can be precisely controlled in time and space, autonomously active soft materials with cell-like responses and spatially controlled structures are promising candidates for a variety of applications, including synthesis of artificial cells having focused functions, soft robotics with directional motion, point-of-care disease diagnosis, and tissue regeneration.

Figure 1 .
Figure 1.Schematic illustration of the research concept.

Figure 2 .
Figure 2. Schematic illustration of cell-like responses and spatiotemporally-controlled structures realized through the key concepts of self-assembly with multiple building blocks.

Figure 3 .
Figure 3. a) Schematic illustration of bidirectional rheological responses of a self-sorting double network (SDN) hydrogel.b, c) Chemical structures and stimulus responses of (b) a peptide-type gelator, NPmoc-F(F)F, and (c) a lipid-type hydrogelator, Phos-cycC 6 .d) Confocal image of the self-sorting double network hydrogel.Green: NPmoc-F(F)F nanofibers, magenta: Phos-cycC 6 nanofibers.e) Rheological analysis of the SDN hydrogels (middle) before and after addition of (left) Na 2 S 2 O 4 and (right) alkaline phosphatase.f) Release rate of embedded proteins from the SDN hydrogels (black) before and after the addition of (blue) Na 2 S 2 O 4 and (red) alkaline phosphatase.Mb: myoglobin, ConA: concanavalin A.

Figure 4 .
Figure 4. a) Schematic illustration of post-assembly fabrication (PAF) of an SDN hydrogel.b) Confocal images of the SDN hydrogels of BPmoc-F 3 and Phos-cycC 6 (left) before and (right) after PAF with sarcosine oxidase and Ca 2+ ions.PCC: Pearson's correlaion coefficient.c) Stimulus response of BPmoc-F 3 through enzymatic generation of H 2 O 2 .d) AND-type gel-to-sol response of the multicomponent SDN hydrogel by addition of sarcosine and ATP.e) Schematic illustration of controlled release of embedded fluorescein-modified IgG (FL-IgG) through the AND-type gel-to-sol response.f) Photographs of the multicomponent SDN hydrogels containing FL-IgG before and after the addition of sarcosine and ATP.g) Release profiles of FL-IgG from the multicomponent SDN hydrogels.

Figure 5 .
Figure 5. a-c) Chemical structures of (a) an enzyme-responsive peptide-type hydrogelator, APmoc-F(CF 3 )F, (b) agarose, and (c) an EAT molecule for avidin.d) Mechanism of enzyme-mediated response toward nonenzymatic proteins.e) Confocal microscopic image of a composite hydrogel of APmoc-F(CF 3 )F and agarose.Magenta: APmoc-F(CF 3 )F nanofibers, green: agarose networks.f) Experimental protocol for controlled release of proteins embedded in the composite hydrogel containing the EAT system, in response to nonenzymatic proteins.g, h) Release rate of (g) myoglobin and (h) RNase A in response to (g) avidin and (h) anti-DNP IgG.

Figure 6 .
Figure 6.a) Schematic illustration of differentiation of a synthetic dipeptide-based coacervate in response to temporal stimulus patterns.b) Chemical structure of a cationic dipeptide derivative, PhePy-FF-OtBu, for liquid-liquid phase separation.c) Scheme for light-triggered radical polymerization under pulsed LED irradiation.d) Confocal images of the coacervates after light irradiation at (top) 9 Hz and (bottom) 0.5 Hz. e) (left) Half-recovery time and (middle) mobile fraction (black) without and with (red) 9 Hz and (blue) 0.5 Hz irradiation.(Right) Frequency-dependence curves of (red) half-recovery time and (blue) mobile fraction.f) Plausible mechanism of temporal pattern-dependent response.

Figure 7 .
Figure 7. a) Schematic illustration of pathway-dependent thermally responsive phase transition of a coacervate.b) Chemical structure of the thermallyresponsive dipeptide derivative.c, d) Confocal images of coacervates at (c) the lower temperature phase (25 °C) and (d) the higher temperature phase (37 °C).e, f) Time-lapse confocal images of thermally-responsive structural changes of the coacervate (e) from 25 to 37 °C and (f) from 37 and 25 °C.

Figure 8 .
Figure 8. a) Schematic illustration of a propagating wave comprising spatiotemporally-coupled formation and degradation of supramolecular nanofibers.b) Reaction network for the propagating wave.c) Time-lapse confocal images of the propagating wave of supramolecular nanofibers.d) Time course of fluorescence intensity at different x-coordinates.

Figure 9 .
Figure 9. a) Reaction scheme for generation of oxime-type peptide hydrogelators, BnOx-F(F)F and CaOx-F(F)F, from the benzaldehyde-tethered peptidetype hydrogelator, Ald-F(F)F.b) Chemical structure of Phos-MecycC 5 .c) Scheme of formation of the interpenetrated self-sorting network through the oxime formation protocol.d) Confocal images before and after the addition of benzylhydroxylamine to a single-network hydrogel composed of Phos-MecycC 5 nanofibers and Ald-F(F)F.Green: peptide-type nanofibers, magenta: Phos-MecycC 5 nanofibers.e) Line plot analysis along a white line shown in (d).f) Time-lapse confocal images of the formation of the peptide-type nanofibers on the surface of the lipid-type nanofibers (highlighted by white arrows) and in the water layer (highlighted by yellow arrows) g) Scheme of formation of the parallel self-sorting network through the oxime exchange protocol.h) (top) Confocal images (left) before and after the addition of (middle) carboxylhydroxylamine and (right) benzylhydroxylamine to the SDN hydrogel composed of Phos-MecycC 5 and Ald-F(F)F.Green: peptide-type nanofibers, magenta: Phos-MecycC 5 nanofibers.(bottom) Line plot analyses along white lines.i) Time-lapse confocal images of the formation of the peptide-type nanofibers on the surface of the lipid-type nanofibers (highlighted by white arrows).

Figure 10 .
Figure 10.a) (top) Chemical structures of agarose and supramolecular hydrogelators.(bottom) Confocal images of the network structures of the composite hydrogels.Green: agarose, magenta: supramolecular nanofibers.b) Proposed mechanism of the network pattern formation.

Figure 11 .
Figure 11.a) Schematic illustration of fracture-induced patterning of the composite hydrogel.b,c) Confocal images of the composite hydrogel of NPmoc-F(F)F and agarose b) before and c) after incubation for 24 h.Green: agarose, magenta: NPmoc-F(F)F nanofibers.d) (top) Photograph of a microneedle stamp with concentric pattern.(bottom) An illustration of the side view of the stamp.e) Confocal images and f) xz-sectional images of the 3D-patterned composite hydrogel punctured with the concentric-patterned stamp.

Figure 12 .
Figure 12. a) Schematic illustration of light-triggered out-of-equilibrium patterning of an SDN hydrogel.b) (left) Scheme for preparation of a lightresponsive peptide-type hydrogelator and its light-responsive isomerization.(right) Chemical structure of Phos-cycC 6 .c) Time-lapse confocal images before and after light irradiation with a line-shaped photomask.Green: peptide-type nanofibers, magenta: lipid-type nanofibers.d) Line plot analysis along a white box shown in (c).e) Plausible mechanism of light-triggered out-of-equilibrium pattering of the SDN hydrogel.