Design Strategies of Stimuli-Responsive Supramolecular Hydrogels

Mar 2, 2017 - Following the detailed structural analysis of a lead hydrogelator that clearly ... early diagnostics and on-demand drug-releasing soft m...
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Design Strategies of Stimuli-Responsive Supramolecular Hydrogels Relying on Structural Analyses and Cell-Mimicking Approaches Published as part of the Accounts of Chemical Research special issue “Stimuli-Responsive Hydrogels”. Hajime Shigemitsu† and Itaru Hamachi*,†,‡ †

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan ‡ Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan CONSPECTUS: Stimuli-responsive hydrogels are intriguing biomaterials useful for spatiotemporal controlled release of drugs, cells, and biological cues, cell engineering for various applications, and medical diagnosis. To date, many physical and chemical stimuli-responsive polymer hydrogels have been developed by chemical modification of polymer chains and cross-linking points. In particular, conjugation with biomolecules to polymers produced promising biomolecule-responsive hydrogels. These examples clearly indicate high potentials of stimuli-responsive hydrogels as promising biomaterials. In addition to polymer hydrogels, supramolecular hydrogels formed by the assembly of small molecules (hydrogelators) via noncovalent interactions have also been regarded as unique and promising soft materials due to their flexible programmability in rendering them stimuli-responsive with the larger macroscopic change (i.e., gel−sol transition). This Account describes our strategies for the rational design of stimuli-responsive supramolecular hydrogels and their biological applications. Following the detailed structural analysis of a lead hydrogelator that clearly indicates the appropriate sites for incorporation of stimuli-responsive modules, we designed supramolecular hydrogels capable of responding to simple physical (thermal and light) and chemical (pH and metal ions) stimuli. More importantly, biomolecule-responsive hydrogels were successfully developed by supramolecularly mimicking the complex yet well-ordered structures and functions of live cells containing multiple components (a cell-mimicking approach). Development of biomolecule-responsive supramolecular hydrogels has been difficult as the conventional strategy relies on the chemical incorporation of stimuli-responsive modules, owing to the lack of modules that can effectively respond to structurally diverse and complicated biomolecules. Inspired by natural systems where functional compartments (e.g., cell organelles) sophisticatedly interact with each other, we sought to integrate the two distinct microenvironments of supramolecular hydrogels (the aqueous cavity surrounded by fibers and the fluidic hydrophobic fiber domain) with other functional materials (e.g., enzymes, peptides or proteins, fluorescent chemosensors, or inorganic porous or layered nanomaterials) for biomolecule responses. In situ fluorescence microscopy imaging clearly demonstrated that chemical isolation and crosstalk are highly successful between the integrated microenvironments in supramolecular hydrogels, similar to organelles in living cells, which allow for the construction of unique optical response and sensing systems for biomolecules. Furthermore, programmed hybridization of our chemically reactive hydrogels with appropriate enzymes can provide an unprecedented universal platform for biomolecule-degradable supramolecular hydrogels. Such biomolecule-responsive hydrogels are a potentially promising tool for user-friendly early diagnostics and on-demand drug-releasing soft materials. We expect that our rational design strategies for stimuli-responsive supramolecular hydrogels by modification of chemical structures and hybridization with functional materials will inspire scientists in various fields and lead to development of novel soft materials for biological applications.

efficient stimuli response of hydrogels to a particular condition or molecule is anticipated to allow for the selective release of a drug or biological cue under biological crude conditions (i.e., in

1. INTRODUCTION Stimuli-responsive hydrogels have attracted much attention not only owing to interests in fundamental science but because of their potential for a wide range of biological and biomedical applications, such as drug delivery systems, regenerative medicines, cancer therapy, and diagnosis.1−4 Selective and © 2017 American Chemical Society

Received: February 3, 2017 Published: March 2, 2017 740

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Figure 1. A combinatorial screening for the development of supramolecular hydrogelators based on solid-phase synthesis.

cells, in culture media, in vivo) and the naked-eye detection of disease biomarkers without expensive analytical instruments. Additionally, a new strategy for cancer therapy by hydrogelation has recently emerged.5,6 Many stimuli-responsive polymer hydrogels have been developed for biological applications.7 Chemical modification of polymer chains and cross-linkers produced various physical and chemical stimuli-responsive polymer-based hydrogels.8 Also, biomolecule-responsive polymer hydrogels have been developed by conjugation with polymers and biomolecules.9−12 These successful examples of stimuli-responsive hydrogels clearly indicate their potential as promising biomaterials. Besides polymer hydrogels, supramolecular hydrogels consisting of small molecules (hydrogelators) have been actively developed and are now recognized as unique and promising soft materials, owing to their high and flexible programmability in lending them stimuli responsiveness.13−16 It is generally accepted that hydrogelators assemble to form nanofibers, which is critical for supramolecular hydrogelation. This fiber formation is driven by a variety of noncovalent interactions such as hydrogenbonding (H-bonding), hydrophobic, van der Waals, and π/π interactions. The delicate balance between these noncovalent interactions can readily modulate the resultant self-assembled structures, which sensitively affects the hydrogelation. The close connection between the gelator structure and macroscopic properties of the resultant hydrogel should enable one to rationally design and tune the properties of stimuli-responsive hydrogels at the small molecule level. In addition, the preparation protocol of supramolecular hydrogels is largely different from that of conventional polymer-based hydrogels that often require polymerization in the presence of adducts such as initiators and

cross-linkers. In the case of supramolecular hydrogels, no adducts are required and the operations (e.g., heating and cooling, simple injection of hydrogelator to aqueous solution, or ultrasound treatment) are quite easy for gelation. This is regarded as an important advantage of supramolecular hydrogelators for biological applications. To date, many supramolecular hydrogels have been reported to exhibit response to stimuli such as heat, pH, metal ions, and light, and these have been applied to cell cultures, control of drug release, and so on. These are usually designed through the insertion of an appropriate stimuli-responsive group into the hydrogelator scaffold. Given the relationship between the gelator structure and the gelation properties, it was reasonably expected that a structural perturbation of the gelator given by a stimulus may cause macroscopic gel−sol or sol−gel changes. However, the employed stimuli have been rather simple so far. The more sophisticated supramolecular hydrogels able to respond to complex biomolecules (i.e., bioactive vitamin, saccharides, DNA, RNA, and noncatalytic proteins) are still limited and difficult to construct,13 while a few enzyme-responsive hydrogels have been reported by several research groups.17−21 A major obstacle for the development of biomoleculeresponsive hydrogels may be the poor molecular recognition capability of synthetic molecules as stimuli-responsive groups, and thus it is difficult to find a suitable module capable of discriminating a target molecule among diverse nontargets in the context of hydrogelator design. Moreover, time-consuming efforts are required for incorporating the modules into the supramolecular hydrogelator, even if stimuli-responsive (molecular recognition) modules with high affinity and selectivity are developed. Therefore, an alternative and more 741

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Figure 2. Self-assembly and hydrogelation by GalNAc-suc-methyl-cycC6 molecules. (a) A schematic representation of hierarchical self-assembly of GalNAc-suc-methyl-cycC6 molecules. (b) Molecular arrangement and noncovalent interactions of GalNAc-suc-methyl-cycC6 molecules in the supramolecular nanofibers. The structure was revealed by X-ray crystallographic analysis. (c) A CLSM image of GalNAc-suc-methyl-cycC6 nanofibers stained by a NBD derivative.

coupling of enzymatic reactions with chemically reactive hydrogelators allowed us to design intelligent supramolecular hydrogels exhibiting unprecedented gel−sol transition in response to disease-related small molecules (biomarkers).34−36

general approach is required for the creation of biomoleculeresponsive supramolecular hydrogels. Based on our successful examples, we herein describe a few strategies for the rational design of stimuli-responsive supramolecular hydrogels composed of lipid mimetic or short peptidebased gelators. One of the strategies substantially relies on the detailed structural analyses of the supramolecular fibers, a fundamental element of supramolecular hydrogels, and the clear characterization of the two different microenvironments inside of the hydrogels. We initially discovered a few supramolecular hydrogelators comprising lipid-like molecules using a combinatorial screening approach.22−26 The molecular packing resolved by X-ray analysis led to rationally modifying the hydrogelators in order to exhibit stimuli-responsiveness for metal ions, pH, and light.27−30 In situ confocal fluorescence microscopy observation also revealed that two distinct microenvironments exist in the supramolecular hydrogels: an aqueous cavity surrounded by self-assembled nanofibers and the hydrophobic domains of their fiber’s interior.31,32 These proved to be useful for optical response/ sensing and chemical (enzymatic) reactions in the hydrogels. Our strategy is also inspired by natural systems such as living cells, an ultimately complex and sophisticated soft material composed of numerous multiple functional components made of protein, lipid, DNA/RNA, etc. Knowledge about living cells has inspired us to hybridize our supramolecular hydrogels with many synthetic or biological molecules bearing various structures and functions. We expected that controlling and facilitating the crosstalk between these multiple components could provide biomolecule responses to the hybrid supramolecular hydrogels. A myriad of organic compounds, metal ions, inorganic materials, biological small molecules, and biopolymers were integrated with the supramolecular hydrogels without loss of function.33 Interestingly, many small molecules are mobile between two (or more) different microenvironments, so that the optical response was effective. Finally, we demonstrated that rational

2. RATIONAL MOLECULAR DESIGN FOR STIMULI-RESPONSIVE SUPRAMOLECULAR HYDROGELS BASED ON LIPID-LIKE MOLECULES It has long been known that some molecules with low molecular weights form hydrogels, and the majority of such hydrogelators were discovered serendipitously.37 In the last two decades, powerful methods for analyzing nanostructures (e.g., electron and atomic force microscopies) revealed that fibrous selfassembled structures and their entangled 3-D networks play an important role in hydrogelation.38 Despite structural analysis, rational design of supramolecular hydrogelators ab initio was difficult. This is because the delicate balance among plural noncovalent intermolecular interactions essential for hydrogelation is unpredictable in aqueous media. Therefore, we first decided to explore a hydrogelator by a combinatorial screening approach using a library of synthetic lipid-like molecules.24 We divided the molecular structure into four modules: a hydrophilic (sugar) head, linker, connector, and hydrophobic tail (Figure 1), and constructed a library of the glycolipid mimics through chemical synthesis. Fortunately, a few hydrogelators were discovered after screening. Spectroscopic analysis and powder X-ray diffraction patterns suggested well-developed H-bonding and van-der Waals packing in a supramolecular hydrogel consisting of glycolipid mimics (Figure 2a). Moreover, as shown in Figure 2b, the detailed X-ray crystallographic analysis confirmed the intermolecular H-bonding networks of the two amides of the linkers and van der Waals interactions of the hydrophobic cyclohexyl (tail) rings in the packing structure.32 Other H-bonding networks were also revealed between the sugar moieties via two interfacial water molecules. Such noncovalent 742

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Figure 3. Rational chemical modifications of a hydrogelator (GalNAc-suc-methyl-cycC6) for development of stimuli-responsive supramolecular hydrogels and the applications of the stimuli-responsive supramolecular hydrogels. (a) A photoresponsive supramolecular hydrogelator (GalNAc-fumcycC6). GalNAc-fum-cycC6 shows cis to trans photoisomerization upon UV irradiation. Therefore, the supramolecular nanofibers are collapsed by UV irradiation, and microscopic gel−sol transition occurs. (b) A photo- and pH-responsive hydrogelator with lysine group (Lys-fum-cycC6). Lys-fumcycC6 molecules interact with each other via intermolecular ion paring and form a stiff photo- and pH-responsive supramolecular hydrogel. (c) A multistimuli responsive hydrogelator with a phosphate group (Phos-fum-cycC6). Phos-fum-cycC6 molecules respond to pH, metal ion, and light in a supramolecular hydrogel in a logic gate fashion.

This detailed structural elucidation allowed us to rationally develop a variety of stimuli-responsive supramolecular hydrogels by the modulation of their noncovalent interactions (Figure 3). We particularly focused on the β-sheet-like well-developed H-bonding networks in the spacer region, as well as the water molecule-bridged saccharide modules at the interface (Figure 2b). It was theorized that the stimuli-induced perturbation of these interactions could substantially affect the stability of the fibers, resulting in a change in the macroscopic hydrogel state. According to this rationale, the incorporation of a photoisomerizable C−C double bond into the connector moiety afforded photoresponsive hydrogels (Figure 3a). UV light irradiation induced a structural change from the trans to cis form at the spacer module, which destabilized the H-bonding network to render the self-assembled fibers to transform into spherical aggregates.28,29 The spherical aggregates were not appropriate for cross-linking each other, relative to the long fibers, so that the gel was destroyed to the sol. Interestingly, the cis form of the spacer region returned back to the trans form by visible light in the presence of Br2, reforming into hydrogel

interactions are thought to cooperatively operate to maintain the well-developed fiber structures. During the study on the gelation mechanism, we sought to use confocal laser scanning microscopy (CLSM) for in situ imaging of the supramolecular fibers without drying processes.31,32 Since we noticed from fluorescence spectroscopy that hydrophobic domains existed in the gelator assemblies, a hydrophobic (and environmentally sensitive) fluorophore, such as nitrobenzoxadiazole (NBD) derivatives, was added to the hydrogel to stain the hydrophobic domains. As shown in Figure 2c, CLSM images clearly demonstrate that there are many well-developed fibers with entanglement and dark spaces surrounded with fiber networks in the wet hydrogel matrix. Using the two distinct microenvironments (i.e., aqueous cavity and the hydrophobic fibers interior) in the hydrogel, these supramolecular hydrogels were successfully applied as functional materials for the thermally controlled release of DNA and for adsorbing bisphenol A, a water pollutant.31 To the best of our knowledge, this is the first example showing the power of CLSM for in situ imaging of supramolecular fibers. 743

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Figure 4. Rational molecular design for stimuli-responsive supramolecular hydrogels consisting of short peptide derivatives. (a) A simple hydrogelator reported by Xu’s group (Fmoc-Y). (b) A strategy for development of stimuli-responsive supramolecular hydrogels. Hydrogelators with chemical reactive groups at the N-terminus are decomposed after addition of a specific stimulus corresponding to each reactive group. (c) Chemical structures of our stimuli-responsive supramolecular hydrogelators and a schematic representation of the gelation and the stimuli-response mechanisms. (d) A chemical structure of a heat-set type hydrogelator.

been shown that very short peptides consisting of only two or three amino acids form hydrogels by appropriate chemical modification.41,42 For example, Xu and Gazit reported a simple Fmoc-Y and Fmoc-FF (Fmoc, fluorenyl-9-methoxycarbonyl; FF, diphenylalanine) as hydrogelators, respectively.17,43 This finding inspired our idea for the rational design of stimuli-responsive hydrogelators comprising short peptides (Figure 4a,b). Xu’s report suggested that a hydrophobic aromatic group, such as Fmoc, at the N-terminus, could play a critical role in stabilizing supramolecular fibers and their networks. We thus assumed that the equipment of a hydrophobic aromatic group removable by stimuli may afford stimuli-responsive supramolecular hydrogels. In design of the H2O2-responsive hydrogel, for instance, dipeptide (FF) was modified at its N-terminus with p-boronophenylmethoxycarbonyl (BPmoc).34 The BPmoc unit reacts with H2O2 through hydroboration reaction, followed by 1,6elimination reaction to be cleaved out along with the generation of p-quinonemethide and CO2 (Figure 4b,c). This chemical reaction-induced structural change diminished the intermolecular interactions crucial for hydrogelation, which led to the destruction of the supramolecular hydrogels. In a similar manner, chemical modification of p-nitro-phenylmethoxycarbonyl (NPmoc) or 6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl (Bhcmoc) unit at the N-terminus of FF peptide derivatives afforded reduction-responsive or photoresponsive supramolecular hydrogels, respectively (Figure 4c). Using dimethylaminocoumarin-4-yl-methoxycarbonyl (DMACmoc) modified FF, a two-photon-responsive supramolecular hydrogel was successfully constructed, which was applied to the control of Brownian motion of nanosized beads and Escherichia coli bacterial movement in the limited micrometer space of the supramolecular hydrogel interior.44 A two-photon response is superior to a one-photon in terms of biological application due to its reduced toxicity and high biocompatibility. Spatiotemporal control of the fluidity inside a soft hydrogel matrix by external

state. Utilizing this photoresponsive gel−sol transition, not only the light-controlled sol−gel patterning of hydrogels but also OFF/ON switching of F1-ATPase rotatory protein motion in a limited small space were carried out. Replacing N-acetylgalactosamine (GalNAc) with lysine in the hydrophilic head of the fiber−water interface yielded a stiff supramolecular hydrogel (G′ > 1.0 × 104) (Figure 3b).39,40 The increased toughness was optimal at the neutral pH region and may be derived from the complementary ion pairs (and/or H-bonding) between the ammonium and carboxylate of the lysine moieties. This hydrogel retained the photoresponsive properties due to the double bond and thus a photofabricated gel mold was prepared for cell culture, differentiation, and 3-D spatial pattering. This hydrogel mold is removable as it is slowly degraded in the culture medium, which offers great promise for biotechnology and regenerative medicine. We further designed and synthesized a multistimuli responsive lipid-like hydrogelator Phos-fum-cycC6 equipped with a phosphate group at the interface, which can bind metal ions (e.g., Ca2+) and with the C−C double bond sensitive to UV light at the spacer (Figure 3c).27 The hydrogel consisting of Phosfum-cycC6 showed four fundamental logic-gate responses exhibiting the macroscopic gel−sol transition by four distinct stimuli such as temperature, pH, Ca2+, and light. This hydrogel also achieved controllable release of bioactive substances in response to UV light, presence of a Ca2+ chelator, and pH changes. We expect that installing logic gate responses to various stimuli in soft materials could be utilized in a variety of applications, such as environment-sensitive actuator, cell culture matrix, and drug-delivery/controlled-release systems.

3. RATIONAL DESIGN OF STIMULI-DEGRADABLE PEPTIDE-BASED HYDROGELATORS A variety of peptide-based gelators for supramolecular hydrogels have been actively developed in recent years. Among them, it has 744

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Figure 5. A cell mimicking approach for development of biomolecule-responsive supramolecular hydrogels. (a) Integration of two distinct environments in supramolecular hydrogels and functional materials for biomolecule sensing. (b) Semiwet enzyme/protein array for optically sensing biomolecules. The sensing mechanism is shown in a blue square.

supramolecular hydrogels, that is, the aqueous cavity and hydrophobic fiber interior. Through the hybridization of a variety of synthetic/biological molecules, we also sought to construct other microenvironments orthogonally located within the hydrogel (Figure 5a). We initially confirmed that the aqueous cavities in supramolecular hydrogels are excellent for the noncovalent immobilization of proteins, peptides, chemosensors, and inorganic materials without loss of their functions. The function of proteins immobilized in the aqueous cavity of supramolecular hydrogels was evaluated using oxygen-bound myoglobin (oxy-Mb), and showed that the half-life of oxy-Mb in the hydrogel (8.7 h) was comparable to or longer than the half-life in water (6.9 h).24 This implies that the supramolecular hydrogels can offer a medium between aqueous fluidic solution and hard dry state, termed “semi-wet environment”, appropriate for the immobilization of natural proteins and enzymes with minimal protein denaturation. The hydrophobic environment, on the other hand, can reversibly entrap hydrophobic molecules in supramolecular hydrogels, which is utilized for a unique fluorescent enzyme (protein/ peptide) array (Figure 5b).32,50,51 When lysyl-endopeptidase (LEP), capable of cleaving a peptide bond of Lys at the C-terminal side, was added to the hydrogel containing a substrate peptide ((Ser)4-Lys-DANSen), the fluorescence of the hydrogel increased and the emission wavelength blue-shifted. This indicates that LEP cleaved the peptide and the released hydrophobic DANSen molecules changed location from the aqueous cavity to the self-assembled nanofibers. This semiwet enzyme/protein array was readily prepared and was potentially applied to quantitative screening inhibitor. In a similar protocol, we prepared a semiwet lectin array using fluorophore-modified lectins (sugar-binding proteins) embedded in supramolecular hydrogels.52 Through the biomolecular fluorescence quenching

stimuli allowed for real-time manipulation of nano- or microsized materials. This strategy was also extended to the design of stimuliresponsive hydrogels exhibiting the sol-to-gel transition, instead of the gel-to-sol transition. We successfully developed a heat-set supramolecular hydrogel by taking advantage of retro-Diels− Alder (r-DA) reaction by designer bolaamphiphies based on short peptide-based hydrogelators (Figure 4d).45,46 Before heating, the gelator was assembled into spheroidal aggregates in the sol state. When the solution was heated at 60 °C for 1 h an entangled 3D network consisting of 1D nanofibers with lengths of several micrometers was observed by TEM, and hydrogelation occurred. This was derived from molecular conversion of bolaamphiphies into hydrophobic hydrogelator by r-DA reaction-triggered removal of the hydrophilic moiety at the N-terminus. Given these results, it is clear that chemical reaction-based modulation of the N-terminal moiety of short peptides is one of the rational design strategies for stimuliresponsive peptide-based hydrogels. Our strategy, that is, tethering a stimuli-responsive (removable) unit, such as arylmethoxycarbonyl (Armoc) groups, at the N-terminus of short peptides, is simple but powerful, which is now followed by many other reports.47−49

4. OPTICAL RESPONSE AND SENSING FOR BIOMOLECULES BY INTEGRATION OF SUPRAMOLECULAR HYDROGEL AND FUNCTIONAL MATERIALS The optical response of supramolecular hydrogels depending on chemical or physical stimuli may provide unique optical sensors composed of soft materials. In order to generate clear optical modulation in response to stimuli (i.e., analytes), we intended to utilize distinct microenvironments embedded in the 745

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Figure 6. A cell mimicking approach for biomolecule-responsive supramolecular hydrogels by using inorganic materials as an artificial organelle. (a) Hydrogels encapsulating polyanion or cation sensing systems composed of supramolecular nanofibers and inorganic porous or layered materials. (b) Polyanion sensing mechanism in the hybrid hydrogels consisting of supramolecular nanofibers, enzyme, dyes, and inorganic porous materials. (c) A photograph of MCM−enzyme supramolecular hydrogel hybrid sensory chip containing a BODIPY derivative (FRET acceptor) after addition of various polyanionic biomolecules. The photograph is taken under UV light.

and recovery (BFQR) method, the fluorescent lectin array carried out pattern recognition of oligosaccharides or cell type. Hybridization of various artificial chemosensors (for phosphorylated-peptides, Zn2+, Ca2+, and pH) with supramolecular hydrogels afforded a noncovalently immobilized sensor array, highlighting that host−guest chemistry can be performed under the supramolecular hydrogel conditions.53−56 Moreover, we constructed more complex sensor systems by incorporation of inorganic nanomaterials and enzymes into the hydrogels (Figure 6a).57,58 Inorganic nanomaterials, such as porous nanoparticles, were expected to provide a third distinct compartment in a hydrogel that may mimic an organelle of live cells. Organelle communication is ubiquitous in nature and known to play important roles in cell growth and proliferation, which leads us to expect that the stimuli-responsive communications between supramolecular fiber and other orthogonal compartments afford a unique sensing capability to the hybrid hydrogels (Figure 6b). Mesoporous silica particles bearing wellordered nanopores, whose surface was functionalized to be cationic (NH2-MCM41), were utilized as colorimetric sensors on the basis of ion-exchange phenomena between encapsulated anionic dyes and polyanions such as carboxylates and phosphates.57 We designed coupling of such anion-exchange ability of NH2-MCM41 to an enzymatic reaction and the hydrophobic supramolecular fiber domain in the semiwet hydrogel matrix. The phosphorylated coumarins (P-coum) encapsulated in the nanopores of NH2-MCMs are kicked out by polyanions having a higher affinity via anion-exchange. The released P-coum is dephosphorylated by phosphatase in the aqueous cavity, and the resultant hydrophobic coumarin moved and condensed in the self-assembled nanofibers. These sequential events were successfully imaged in situ by CLSM measurements. As a result, supramolecular hydrogels changed their optical properties depending on the added anions so that

the sensor array can discriminate polysulfates from polyphosphates (Figure 6c). Montmorillonite (MMT), layered inorganic clay with negative charges, was also embedded in the supramolecular hydrogel as a host of cationic fluorescent dyes (Figure 6a).58 In this case, polycations caused an ion-exchange reaction to facilitate the release of the cationic fluorophore from MMT. The released fluorophore moved to the hydrophobic fiber domain to enhance the fluorescence intensity with a blue-shift of wavelength. This polycation-triggered fluorescence modulation of the MMT/hydrogel hybrid allowed for optical detection of biological polyamines, such as spermidine and spermine, in artificial urine. It is clear that orthogonal domains given by these materials and coupling with enzymatic reactions in supramolecular hydrogels efficiently render the hybrid hydrogels semiwet sensors with unique optical properties and selectivity.

5. BIOMARKER-DEGRADABLE HYDROGELS BY INTEGRATION OF REDOX-ACTIVE SELF-ASSEMBLED NANOFIBERS AND ENZYMES Hybridizing various enzymes with chemically reactive supramolecular hydrogels enabled the programmable design of sophisticated soft materials responsive to structurally and chemically complicated small biomolecules.34,35 There are biomolecules such as vitamins, lipids, proteins, DNA, and RNA, whose changes in concentration and expression levels are closely related to their corresponding physiological disorder and pathology. Some biomolecules exhibiting high diversity and complexity in structure and functions are called biomarkers. The development of biomarker-responsive hydrogels remains challenging, although it is anticipated to be a promising soft material. With the aim of construction of such unprecedented hydrogels, we sought to employ natural enzymes bearing the rigid substrate selectivity together with chemically reactive supramolecular hydrogels such as H2O2-responsive hydrogel 746

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Figure 7. Biomolecule degradable supramolecular hydrogels consisting of stimuli-responsive hydrogels and enzymes. (a) Biomolecule-responsive supramolecular hydrogels consisting of H2O2-responsive gel fibers and oxidases. Oxidases generate H2O2 as a byproduct by enzymatic reaction, and the generated H2O2 decomposes the gel fibers. Finally, the gel changes to sol. (b) Serial coupling of enzymatic reactions in the BPmoc-FFF hydrogel for expansion of a chemical-stimuli response.

Figure 8. Supramolecular hydrogels in response to two biomolecules. (a) Lactic acid and NADH responsive supramolecular hydrogels consisting of reduction-responsive hydrogel (NPmoc-FF), LDH, and NR. An optical photograph in the figure shows the results of gel−sol response test. (b) Boolean logic gate (AND) response of the supramolecular hydrogel for glucose and NADH. The hydrogels are composed of hybrid supramolecular nanofibers of BPmoc-FFF and NPmoc-FF, GOx, and NR. G and S indicate gel and sol, respectively. 747

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Figure 9. Schematic representation of self-sorting between peptide- and lipid-type hydrogelators and in situ CLSM imaging of the self-sorted nanofibers. Scale bar: 5 μm.

(BPmoc-FF).34 It is well-known that many oxidases are able to oxidize the corresponding substrates (biomarkers in some cases) with the concurrent generation of H2O2 as a byproduct. Therefore, we expected that H2O2 produced upon enzymatic reaction was able to decompose the BPmoc-FF hydrogel matrix to cause the gel−sol transition, when an oxidase is embedded in the hydrogel. Indeed, the addition of glucose to BPmoc-FFF (the hydrogelator superior to BPmoc-FF in terms of critical gelation concentration (CGC) 0.05 wt %) hydrogels containing glucose oxidase (GOX) induced the gel−sol transition, as shown in Figure 7a. Owing to the rigid substrate selectivity of GOX, this hybrid gel did not respond to galactose or lactose. In a similar protocol, we prepared BPmoc-FFF hydrogels encapsulating various oxidases (sarcosine (SOX), choline (COX), urate oxidases (UOX)) and revealed that each hybrid hydrogel selectively responded to substrates of the corresponding oxidases (Figure 7a). It is note-worthy that glucose (diabetes), sarcosine (prostate cancer), and uric acid (gout) are known as biomarkers, and this biomarker-responsive gel−sol response is easily visible by the naked eye. The concurrent encapsulation of two enzymes in the hydrogel allows for increasing the variety of analytes (Figure 7b). When GOX was embedded together with glycosidase in BPmoc-FFF, the addition of lactose to the hybrid hydrogel caused a gel−sol transition. This indicated that the cascade reaction had occurred; thus, lactose was hydrolyzed by glycosidase to generate glucose and galactose. The resultant glucose was subsequently oxidized by GOX to generate H2O2, which destroyed the BPmoc-FFF hydrogel. The similar cascade reaction took place by choline esterase and COX in the hydrogel matrix, which enabled the detection of acetylcholine by the gel−sol transition. In the case of NPmoc-FF gel, on the other hand, we are able to hybridize nitroreductase (NR) and NADH (NAD) as its cofactor (Figure 8a). Combining NR to lactate dehydrogenase (LDH) and NAD in the NPmoc-FF gel matrix afforded lactic acid responsive supramolecular hydrogels. Lactic acid was oxidized by LDH to generate NADH from NAD, which reduced nitrophenyl group of NPmoc-FF to the gel decomposition. Furthermore, we succeeded in building Boolean logic gates (OR and AND) in the hybrid hydrogel containing multiple components (Figure 8b). For instance, the mixed hydrogel composed of BPmocFFF and NPmoc-FF containing GOX and NR was able to simultaneously sense plural biochemicals (glucose and NADH) and execute a controlled drug release in accordance with the logic operation. Molecular logic-gate is actively explored.59 However, the

employed input signals were somewhat simple, such as pH, cations, or anions, and outputs are largely limited to physical signals such as fluorescence or electric signals in most cases. It is conceivable that the present example sidesteps these limitations by rational coupling of chemically reactive supramolecular hydrogels with natural enzymes. We further revealed that a signal amplification circuit originally developed by Shabat was useful as a cross-talking component embedded in the enzyme-hybrid hydrogel, leading to enhance the detection limit of biomarkers.60,61 Since both the oxidase reaction with the substrates and the subsequent chemical reactions of H2O2 with BPmoc-FFF are stoichiometric, that is, a 1:1:1 substrate/H2O2/BPmoc-FFF stoichiometry exists, biomarker sensitivity was strictly limited. In case the detectable concentration of a biomarker is higher than the critical value for a specific disease, the sensitivity should be improved. We optimized an amplification system (a pair of synthetic amplifier and SOX) for the BPmoc-FFF hydrogel, which produced 2 mol of H2O2 from 1 mol of substrate (H2O2). In practice, we demonstrated that a multicomponent BPmoc-FFF hydrogel containing the synthetic amplifier/SOX/UOX successfully created a userfriendly, naked eye detection sensor for the disease level of uric acid in human plasma.

6. CONCLUSION AND FUTURE DIRECTIONS We briefly present a few promising strategies for the rational design of stimuli-responsive supramolecular hydrogels. Deep understanding of both the intermolecular interactions of the gelators and the resultant microenvironment is crucial for achieving the designed gel−sol/sol−gel transition or optical changes. In addition, it should be emphasized that integration of supramolecular hydrogels with various functional materials such as fluorescent probes, chemical sensors, natural enzymes/proteins, and inorganic materials allowed us to obtain unique responses to complicated biomarkers in supramolecular soft materials. Very recently, we directly imaged with super-resolution CLSM a hydrogelator pair (BPmoc-FFF and Phos-fum-cycC6) dynamically self-sorting and orthogonally assembling into two distinct supramolecular nanofibers in situ (Figure 9).62 Such a multicomponent but well-ordered structure is reminiscent of the cytoskeleton of living cells. Supramolecular chemistry, a field originating from the strong interest in remarkably well-ordered structures and intelligent functions of biomolecules, has made great efforts for mimicking these biomolecules or systems. 748

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Accounts of Chemical Research

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However, there remains a large gap between artificial supramolecular systems and natural (sophisticated) ones. Natural systems consisting of multiple components can effectively regulate entropy, fluctuation, and chemicals as energies under very crude conditions and in far-from-equilibrium states, while most artificial systems are rather simple in the pure and equilibrium state. We believe that by controlling multicomponent supramolecular assemblies in their nonequilibrium, orthogonal or cooperative, and spatiotemporal manners we can generate novel smart materials. These efforts may also open up “complex (crude) systems chemistry” beyond biomimetics.

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AUTHOR INFORMATION

Corresponding Author

*Itaru Hamachi. E-mail: [email protected]. ORCID

Itaru Hamachi: 0000-0002-3327-3916 Notes

The authors declare no competing financial interest. Biographies Hajime Shigemitsu received his Ph.D. from Osaka University under the supervision of Prof. Mikiji Miyata in 2013. He carried out his postdoctoral research in the group of Prof. Itaru Hamachi at Kyoto University. His research interests include supramolecular chemistry and functional materials chemistry. Itaru Hamachi obtained his Ph.D. at the Department of Synthetic Chemistry of Kyoto University in 1988 under the supervision of Prof. Iwao Tabushi. He started his carrier in the field of supramolecular chemistry at Kyushu University in 1988 and then shifted his research field to protein engineering as an associate professor there. In 2001, he became a full professor at Kyushu University and then moved to the Department of Synthetic Chemistry and Biological Chemistry of Kyoto University in 2005. His interest has now been extended to chemical biology and organic chemistry in living systems and supramolecular biomaterials.

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ACKNOWLEDGMENTS We thank all former and current members of the Hamachi laboratory who have contributed to the described work. The work described herein was supported by generous funding from Japan Science and Technology Agency (JST), Japan Society for Promotion of Science (JSPS), and the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). H.S. acknowledges a JSPS research fellowship for young scientists.

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REFERENCES

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DOI: 10.1021/acs.accounts.7b00070 Acc. Chem. Res. 2017, 50, 740−750

Article

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DOI: 10.1021/acs.accounts.7b00070 Acc. Chem. Res. 2017, 50, 740−750