DOI: 10.1002/asia.201500563
Focus Review
Supramolecular Chemistry
Supramolecular Assemblies Responsive to Biomolecules toward Biological Applications Hajime Shigemitsu[a] and Itaru Hamachi*[a, b]
Chem. Asian J. 2015, 10, 2026 – 2038
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Focus Review Abstract: Stimuli-responsive supramolecular assemblies consisting of small molecules are attractive functional materials for biological applications such as drug delivery, medical diagnosis, enzyme immobilization, and tissue engineering. By use of their dynamic and reversible properties, many supramolecular assemblies responsive to a variety of biomolecules have been designed and synthesized. This review focuses on
1. Introduction Much attention has been paid to stimuli-responsive supramolecular assemblies as one of the smart materials in various fields.[1] Among them, supramolecular assemblies responsive to biomolecules are promising as drug-delivery carriers, reagents for detection and imaging of biomolecules, and others.[2] Albeit being in an early stage, a myriad of attempts toward biomedical application using supramolecular materials have been made in recent years. To date, nano-sized biomaterials made of organic polymers and inorganic nanoparticles have been mainly studied because their mechanical toughness and stability are crucial for biological applications.[3] In particular, drug- or gene-delivery vehicles composed of polymers have been remarkably improved in their functions in the last decade.[4] On the other hand, supramolecular assemblies consisting of small molecules have not been well developed in this research direction yet. Neither guidelines for their molecular design nor methods for controlling their aggregation morphologies suitable for biological application have been established. However, the designability of stimuli-responsiveness, reversibility, homogeneity, and finely tunable properties are intriguing in supramolecular assemblies composed of small molecules. With the aim of a rational design of supramolecular assemblies as unique biomaterials, it is apparent that fundamental understanding of the assembly behavior in water and, more favorably, under crude conditions is keenly needed. One of the important features of supramolecular assemblies represents stimuli-responsiveness, which is a key for producing intelligent biomaterials. Installing a response to biomolecule into supramolecular materials would allow for various applications such as drug delivery, medical diagnosis, and imaging and monitoring of specific biomolecules. So far, many researchers have employed supramolecular assemblies composed of peptides and/or amphiphilic lipid derivatives and sought to [a] Dr. H. Shigemitsu, Prof. Dr. I. Hamachi Department of Synthetic Chemistry and Biological Chemistry Graduated School of Engineering Kyoto University Katsura, Kyoto 615-8510 (Japan) E-mail: [email protected] [b] Prof. Dr. I. Hamachi Core Research for Evolutional Science and Technology (CREST) Japan Science and Technology Agency (JST) 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075 (Japan) Chem. Asian J. 2015, 10, 2026 – 2038
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promising strategies for the construction of such dynamic supramolecular assemblies and their functions. While studies of biomolecule-responsive supramolecular assemblies have mainly been performed in vitro, it has recently been demonstrated that some of them can work in live cells. Supramolecular assemblies now open up new avenues in chemical biology and biofunctional materials.
reveal the correlations between the molecular structures and their aggregation/response behaviors.[5] Further progress in unveiling these complicated relationships should lead to the rational design of stimuli-responsive supramolecular assemblies. In addition, intensive studies of dynamic supramolecular assemblies in multi-component systems would contribute not only to creating novel biomaterials but also to shedding light on behaviors of synthetic molecules in living cells. In nature, various molecules cooperatively or orthogonally interact and play roles in maintaining homeostasis and adapting to environmental changes. It is considered that simple supramolecular assemblies comprising multiple components should be a good model of such complex systems, which may lead to the synthesis of cell-mimetic supramolecular systems In this Focus Review, we describe the recent progress in biomolecule-responsive supramolecular assemblies consisting of small molecules, particularly by focusing on micelles, vesicles, and nanofibers (hydrogels). Although these supramolecular materials have been mainly studied in vitro so far, a few recent examples clearly showed that supramolecular assemblies can maintain their aggregation structures and function even under cellular conditions, implying that supramolecular assemblies exhibit much potential for biological applications in cells or in vivo. We start this review with examples of supramolecular assemblies that are responsive to small biomolecules through chemical reactions in vitro. Next, supramolecular assemblies responsive to small-molecule recognition, enzymatic reactions; and protein recognition are sequentially described. Finally, successful applications of several supramolecular assemblies in living cells are summarized.
2. Supramolecular Assemblies Responsive to Small Biomolecules via Chemical Reaction In the first section, we introduce supramolecular assemblies responding to small biomolecules through chemical reactions. Most of such assemblies reported so far are categorized into supramolecular gels. Such gels may be potentially applied in the rapid and convenient diagnosis of biomarkers, since the gel–sol transition is easily detectable by the naked eye. In addition, the controlled drug release may be another attractive application due to the possibility of encapsulating various molecules in the gels. In living cells, there are many small molecules such as various cations and anions, reactive oxygen species (ROS), gluta-
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Focus Review thione (GSH), adenosine triphosphate (ATP), sugars, lipids, and so on. Such small molecules play important roles to maintain homeostasis of living systems. For instance, GSH, a simple tripeptide, is known to control the reductive environment in live cells. ATP is a key energy source for various enzymatic reactions and mechanical motions of proteins. Utilization of such molecules as biological stimuli is expected as a rational strategy for designing bio-responsive materials. Although many synthetic polymers responsive to biomolecules have been reported,[6] supramolecular assemblies exhibiting such functions have been limited. Yang and co-workers showed the first example of GSH-triggered hydrogelation of an oligopeptide scaffold containing a disulfide bond (Figure 1).[7] The designer oligopeptide Nap-
struct supramolecular gels responsive to small molecules via chemical reactions was proposed, which relied on tethering appropriate chemically reactive groups to the N-terminus of short peptides (Figure 2 a).[10] For example, modification of an oligo-phenylalanine peptide with phenylboronic acid 3 at the N-terminus successfully afforded H2O2-responsive hydrogels (Figure 2 b). The appended phenylboronic acid reacts with H2O2 and the subsequently induced 1,6-elimination reaction destroys the molecular structure of the hydrogelator to cause the gel–sol transition. In the cases of nitrobenzene- and hydroxycoumarin-modified peptides (4 and 5), the gel–sol transitions were triggered by reductive reagents (e.g., Na2S2O4, NADH) and photo-irradiation, respectively (Figure 2 b). The Hamachi group constructed supramolecular hydrogels encapsulating several enzymes as new biomarker-responsive gels.[11] The hybrid gels composed of a H2O2-responsive hydrogelator and an oxidase which produces H2O2 as a by-product during substrate oxidation result in gel–sol transition in the presence of a biomarker (i.e., a substrate of an oxidase). This system is applicable for various oxidases and thus provides a new method to expand the responsiveness and possibility as a smart supramolecular gel for easy diagnosis. Yang and colleagues achieved a reversible gel–sol transition system by a redox reaction inspired by natural redox systems (e.g., NADH/NAD) (Figure 3).[12] The peptide containing a selenium atom (7) assembles fibrously and forms supramolecular hydrogels. Upon oxidation by H2O2, the peptide 7 changes to 6 and the self-assembled fibers convert into micelles (Figure 3 b– d). The resultant selenoxide compound 6 could be reduced by
Figure 1. (a) Molecular structures of oligopeptides 1 and 2. (b) Optical images showing hydrogelation after addition of GSH. (c,d) TEM images of self-assembled structures of (c) 2 and (d) 1. Adapted from Ref. [7] with permission from The Royal Society of Chemistry.
Itaru Hamachi obtained PhD 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 of 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.
GFFYE-CS-EERGD 2 is composed of three functional parts (Figure 1 a). The Nap-GFFYE part 1 constructs supramolecular fibers and gelates water, and the EERGD part guarantees a sufficient solubility in water. The disulfide bond (CS unit) connects the two peptide parts, which can be cleaved by GSH to give Nap-GFFYE and hydrogels (Figure 1 b). TEM observations of self-assemblied structures of 1 and 2 show a fibrious and an amorphous structure, respectively (Figure 1 c,d). The GSH-inductive hydrogel does not need any heating operation for gelation, which is favorable for biological applications. The group confirmed the biocompatibility of the gels,[8] suggesting a potential for regenerative medicine and cancer therapy. In the intracellular space, high levels of GSH maintain a reductive environment; by contast, GSH is present at low levels in the extracellular matrix. Therefore, utilizing GSH is a rational development for supramolecular assemblies working only inside living cells.[9] Gel–sol transitions, but not sol–gel transitions, by small biomolecules have also been reported. A rational design to conChem. Asian J. 2015, 10, 2026 – 2038
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Hajime Shigemitsu received his PhD 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 material chemistry, supramolecular chemistry and chemical biology.
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Figure 2. (a) Scheme showing the self-assembly process of peptide-based hydrogelators to form supramolecular hydrogels and their stimuli-responsive gel– sol transition. (b) Stimuli-responsive degradation mechanisms of 3, 4, and 5. Adapted from ref. [10].
Figure 3. (a) Chemical structures of selenium-containing peptides (6 and 7) and optical images of the solution and the gel. (b) Schematic representation of the redox-triggered transformation between micelles of 6 and nanofibers of 7. (c,d) TEM images of the micelles of 6 (c) and nanofibers of 7 (d). Adapted from ref. [12].
vitamin C to restore the original peptide structure and recover the hydrogel state again. Interestingly, the rather small change between selenide and selenoxide critically affects the assembling behavior so as to successfully give the reversible gel–sol transition by biological stimuli.
3. Supramolecular Assemblies Responsive to Molecular Recognition Owing to the relatively weak non-covalent interactions between components of supramolecular assemblies, it is expected that they easily change their morphology and optical property by interacting with guest molecules. Since molecular recognition does not destroy molecular structures unlike chemical reactions, the response relying on molecular recognition may afford reversibly responsive biomaterials. In this section, some of the pioneering works are discussed. Chem. Asian J. 2015, 10, 2026 – 2038
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3.1. Supramolecular Assembly or Disassembly Triggered by Molecular Recognition In 2003, Xu and colleagues reported stimuli-responsive supramolecular hydrogels using ligand–receptor interaction.[13] They exploited a simple hydrogelator 8 and a strong interaction between the d-Ala-d-Ala peptide sequence and vancomycin,[14] a well-known antibiotic, to induce gel–sol transition (Figure 4). This is the first achievement with regard to gel–sol transition triggered by molecular recognition. Schneider and co-workers reported zinc-triggered hydrogelation of self-assembling b-hairpin peptide 9 (Figure 5).[15] Insertion of 3-amidoethoxyaminodiacetoxy-2-aminopropionic acid into the b-hairpin peptide gives a hydrogelator showing the formation of Zn2 + -triggered supramolecular nanofibers and hydrogelation (Figure 5 c). Zn2 + plays an important role in inducing folding and the self-assembly of peptide 9 (Figure 5 a). The control peptide 10 that lacks the chelator part does not display such Zn2 + -responsiveness. Zinc is a crucial metal for life, and
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Focus Review 3.2. Molecular Recognition without Altering the Assembly and the Optical Sensing of Small Biomolecules
Figure 4. (a) Molecular structure of hydrogelator 8 and (A) its hydrogel. The phase transitions of the hydrogel induced by the addition of (B) 0.01, (C) 0.1, and (D) 1.0 equiv of solid vancomycin into the hydrogel. (b) Schematic representation of ligand–receptor interactions between vancomycin and the gelator. The interaction induces the gel–sol phase transition. Adapted from ref. [13]. Copyright 2003 American Chemical Society.
Figure 5. (a) Proposed mechanism of metal-triggered folding and self-assembly of 9. (b) Primary sequences of gelator 9 showing Zn2 + -responsiveness and the control peptide 10. (c) Optical images of the gel and solution of gelator 9. Adapted from ref. [15].
Zn2 + ions promote matrix metalloproteinase debridement and keratinocyte migration to accelerate wound repair. The amount of Zn2 + present in the hydrogel network is controlled by the amount of self-assembling b-hairpin peptide. Zn2 + would be deliverable to a wound bed using such a gel system. In another example of metal-ion-induced hydrogelation, a lipid-like molecule having a phosphate group shows gelation by addition of Ca2 + ions.[16] Observation by confocal laser scanning microscopy (CLSM) revealed that Ca2 + ions bridge nanofibers and enhance the strength of the fibril networks. Host–guest interactions are also employed for the construction of supramolecular vesicles. Liu and co-workers reported that complexation of ATP with an amphiphilic calixarene 11 produces hollow spherical nanoparticles (Figure 6).[17] The critical micellar concentration of the calixarene 11 largely decreased by ATP binding due to charge neutralization. Interestingly, the hydrolysis of encapsulated ATP by phosphatase destroys the self-assembled vesicles. The assembly and disassembly of such vesicles, depending on ATP encapsulation, took place reversibly. Chem. Asian J. 2015, 10, 2026 – 2038
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In some supramolecular assemblies, only their optical properties were altered upon binding of guest molecules without any morphological changes. Kimizuka and co-workers utilized the vesicle surface for luminescent ATP sensing (Figure 7).[18] The vesicle composed of amphiphilic TbIII complex 12-TbIII senses ATP and ADP by exchange of ligands coordinated to TbIII from H2O to phosphate groups. The ligand exchange causes a sigmoidal increase in luminescent intensity due to the difference in the energy transfer from ligands to TbIII. The high density of cationic TbIII complex on the surface allowed the sensitive detection of low concentrations of ATP. In another example, it was reported that sugar-based hydrogels can be used to detect low concentrations of insulin through changes in the fluorescent intensity, without changes in their fibrous morphology.[19] Chiroptical properties of selfassembly induced by ATP derivatives were successfully used to monitor enzymatic ATP hydroly-
Figure 6. Schematic illustration of the self-assembly of 11 with ATP and its phosphatase response. Adapted from Ref. [17] with permission from The Royal Society of Chemistry.
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Focus Review materials, and enzymes can be concurrently embedded in supramolecular gels while retaining their orginal activities and functions. For instance, supramolecular gels hybridized with fluorescent dyes containing inorganic materials (Montmorillonite (MMT) or mesoporous silica particles (MCM41)) can work as a fluorescent sensor for biologically important polyamines or polyanions (Figure 9).[23, 24] These results also revealed the new possibility of generating supramolecular gels consisting of multiple components.
4. Supramolecular Assemblies Responsive to Enzymatic Reactions Figure 7. Schematic representation for self-assembly of amphiphilic 12-TbIII and binding of ATP molecules. Adapted from ref. [18]. Copyright 2011 American Chemical Society.
sis (Figure 8).[20] The supramolecular polymer consisting of a naphthalene bisimide derivative equipped with dipicolylamine Zn2 + complex 13 constructs a P-type supramolecular helix upon ATP binding (Figure 8 a). On the other hand, ADP or AMP complexation induced an M-type helix. This chiral inversion phenomenon is available for monitoring the enzymatic activity (Figure 8 b). Fluidic circumstances formed by supramolecular gels provide a unique matrix for sensing various small biomolecules.[21, 22] Hamachi and co-workers demonstrated that various functional materials such as dyes, layered and porous inorganic
Some particular enzymes are overexpressed in disease-related cells and tissues, and thus supramolecular assemblies responsive to such enzymatic reactions could lead to chemotherapeutic and diagnostic tools. Given that enzymes catalyze the formation or cleavage of chemical bonds with high selectivity, the incorporation of a module susceptible to an enzymatic reaction into a component of supramolecular assemblies is a promising strategy for rational design to produce a supramolecular assembly responsive to a specific enzyme. 4.1. Gelation and Gel–Sol Transition Triggered by Enzymatic Reactions
Figure 8. (a) Molecular structure of NDPA 13 along with a pictorial representation of adenosine phosphates (APs) inducing a helical supramolecular organization. (b) Schematic illustration of the dynamic helix reversals upon enzyme (CIAP) action on the NDPA-bound ATP molecules and their respective real-time chiroptical readout. Adapted from ref. [20]. Copyright 2014 Nature Publishing Group. Chem. Asian J. 2015, 10, 2026 – 2038
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Xu and colleagues reported supramolecular hydrogel formation triggered by an enzymatic reaction in 2004 (Figure 10).[25] Dephosphorylation of a simple amino acid derivative by a phosphatase resulted in a hydrogelator and subsequent formation of hydrogels. Conversion of an anionic group into a neutral group can effectively change the balance of the hydrophobicity and hydrophilicity to yield a hydrogelator. After this report, various hydrogelators generated by many types of enzymatic reactions (kinase, lactamase, peptidase, and esterase) have been developed.[26]
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Focus Review Hydrogelation by enzymatic reactions is applicable for improvement of drug dispersity. The group of Xu synthesized a hydrogelator precursor containing taxol, which is a potent anticancer drug in spite of its poor solubility and dispersibility in water.[27] The enzyme-triggered hydrogelation yielded supramolecular hydrogels containing taxol without loss of its potency. 4.2. Multicomponent Systems of Supramolecular Assemblies Responsive to Enzymatic Reactions
Figure 9. Hybrid materials of supramolecular gels and inorganic materials (a: MCM41, b: MMT). (a) Chemical structures of compounds used in this work (Suc-8S, IP6, heparin, and chondroitin sulfate are the biomolecules sensed in this system), and construction and mechanism operating in the fluorescent dye-encapsulated MCM/enzyme/ supramolecular hydrogel hybrid sensory system. (b) Chemical structures of compounds used in this work (spermine and spermidine are the anionic biomolecules detected by this system), and construction and the mechanism of action of the fluorescence dye (G-coum) adsorbed MMT/supramolecular hydrogel 18 hybrid sensory system for polyamines. Adapted from ref. [23] and [24]. Copyright 2009 and 2011 American Chemical Society. Chem. Asian J. 2015, 10, 2026 – 2038
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Multicomponent systems of supramolecular assemblies are also intriguing for the design of enzyme-responsive supramolecular assemblies. Huang and colleagues demonstrated enzymetriggered supramolecular assemblies by a host–guest system between cyclodextrin (CD) and surfactants.[28] While these host– guest complexes do not aggregate in water, amylase decomposes CDs to release the corresponding surfactants that form supramolecular assemblies. Liu and co-workers reported enzyme-triggered disassembly of vesicles consisting of host–guest complexes of calixarene derivatives and myristoylcholine.[29] The embedded drugs can be released when these vesicles are destroyed by cholinesterase capable of hydrolyzing myristoylcholine.[30] van Esch and co-workers developed a three-component hydrogel consisting of supramolecular fibers, vesicles, and an enzyme (chymotrypsin).[31] Interestingly, the supramolecular fibers of 21 and the vesicles containing the enzyme orthogonally exist in the hydrogel matrix (Figure 11). The enzyme molecules encapsulated in the vesicles could decompose the gel fibers only when they are re-
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Focus Review metalloproteinase-2 (MMP2) assembled to form nanoparticles (Figure 12 c).[32, 33] In the nanoparticles, the 19F NMR signal is not detected due to the increase in their apparent molecular weight. After collapse of the nanoparticles by MMP2, the 19F NMR signal sharply recovered (FigFigure 10. (a) Chemical structures of the hydrogelator precursor 19 and hydrogelator 20. (b) Optical images of soure 2 b). This turn-on 19F NMR lution of 19 and gel of 20. 19 is hydrolyzed by phosphatase to give 20 and hydrogels. Adapted from ref. [25]. signal-detection system was successfully applied to monitoring the MMP2 activity. Aggregation-induced emission (AIE) is exploited for fluoresleased. The release rate of the enzyme can be tuned by the heating temperature and time, and thus precise control of the cent monitoring of an enzymatic activity. Cationic myristorylgel–sol transition is achieved. choline and anionic tetraphenylethylene (TPE) derivatives interacted with each other in water and form nanoparticles.[34] The resultant nanoparticles show a strong emission due to AIE of 4.3. Optical Sensing and Monitoring of Enzymatic Reactions TPE. After the nanoparticles are destroyed by choline esterases, by Supramolecular Assemblies the emission is reduced, thus enabling monitoring of the cholineesterase activity in a turn-off mode. Turn-on specific detection of enzymatic activities is of fundamental importance in drug discovery and medical diagnosis. Several self-assembling nano-aggregates are applicable to turn-on type imaging of enzymatic activity. Hamachi and co5. Supramolecular Assemblies Responsive to workers reported that amphiphilic molecules 22, 23, and 24 Biopolymers other than Enzymes having a 19F-modified peptide sequence cleavable by matrix There are various biopolymers (e.g., proteins, DNA, RNA, and oligo- or polysaccharides) other than enzymes in cells. Some of them are closely related to various diseases, which makes them important targets to be responded to or to be detected. However, it is much more difficult to design supramolecular assemblies that can respond to such biopolymers, compared to enzymes. This is because these biopolymers simply exhibit
Figure 11. (a) Chemical structure of gelator 21 bearing fluorogenic 6-AQ. (b) Self-assembled multicomponent system: a solution of 21 in DMSO is added to an aqueous solution of the enzyme chymotripsin loaded in liposomes. Heating the gel results in partial liberation of the enzyme into the gel matrix, allowing for release of 6-AQ through enzymatic hydrolysis of the amide bond. Adapted from ref. [31]. Copyright 2012 American Chemical Society. Chem. Asian J. 2015, 10, 2026 – 2038
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Figure 12. (a) Chemical structures of self-assembled probes. (b) Schematic illustration of a substrate-based self-assembling 19F probe for turn-on detection of enzymatic activity. (c) Fluorescent microscopy image of probe 22 (50 mm) loaded with Nile Red. Scale bar: 5 mm. Adapted from ref. [32].
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Figure 13. (a) Chemical structure of Nap-GFFYGGGWRESAI (nongelator) 25 with a possible self-assembly ability. (b–d) Nap-GFFYGGGWRESAI assembles into nanofibers which do not interact in aqueous solution, thus resulting in fiber networks with low density of cross-linking points. (e–g) The addition of rationally designed fusion protein (ULD-TIP-1) enhances the interactions between fibers, thus leading to hydrogelation. Scale bars: 500 nm. Adapted from ref. [35].
a molecular recognition capability, but do not catalyze any chemical reactions. The construction of such supramolecular assemblies requires an elaborate control of non-covalent interactions depending on these biopolymers. Yang and co-workers reported hydrogel formation triggered by protein recognition.[35] They used a fusion protein (ULD-TIP1) as a glue between supramolecular nanofibers of 25 containing an oligopeptide recognized by TIP-1 (Figure 13). ULD-TIP1 is composed of a ubiquitin-like domain (ULD) from the special AT-rich sequence binding protein-1 (SATB-1) and Tax-interaction protein-1 (TIP-1). Since ULD proteins predominantly form a tetramer, ULD-TIP-1 crosslinks the supramolecular nanofibers to afford hydrogels upon TIP-1 interaction with the fibers. The obtained gels showed thixotropic fast gelation properties (Figure 13 e,f,g). It is well known that proteins interact with specific small molecules termed protein ligands. The ligand–protein recognition is selective and thus useful for the development of dynamic biomaterials. In 2009, Hamachi and co-workers found that self-assembled nanoparticles consisting of ligand–probe conjugates can selectively respond to a protein and thus are applicable to protein sensing (Figure 14 ).[36] A molecule containing a protein ligand and fluorine atoms spherically self-assembles and shows no 19F NMR signal due to an increase in the apparent molecular weight. The 19F NMR signal was recovered by disassembly of these nanoparticles upon interaction of a target protein with the ligand part of nanoparticles. This assembly/disassembly principle induced by protein recognition was extended from 19F NMR (MRI) to fluorescent detection. Various ligands and fluorescent dyes such as boron dipyrromethene (BODIPY), fluorescein, and tetramethyl rhodamine (TMR) Chem. Asian J. 2015, 10, 2026 – 2038
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Figure 14. Off/on 19F NMR probes for protein imaging. (a) Strategy used for an off/on switching system of 19F NMR. (b) Chemical structures of the probes with various ligands. Adapted from ref. [36]. Copyright 2009 Nature Publishing Group.
are available for this detection system.[37, 38] This study also highlighted the significance of disassembly of supramolecules, in addition to providing a new sensing strategy for protein detection of proteins. Ajayaghosh and colleagues reported self-assembled nanoparticles composed of near-IR squaraine dyes that specifically reacted with human serum albumin (HSA).[39] The nanoparticles can sense serum albumin protein (SAP) even in the presence of other competing thiol-containing proteins and small molecules. Thayumanavan and co-workers constructed a supramolecular assembly equipped with logic-gate-like responsiveness for enzymatic reaction and protein recognition.[40] In contrast to protein-responsive supramolecules, dynamic supramolecular assemblies responsive to DNA, RNA, and saccharides have been poorly developed to date. These should be explored in the near future.
6. Application of Supramolecular Assemblies in Cells and in Vivo Encouraged by successful examples of supramolecular assemblies responsive to biomolecules in vitro, several groups recently sought to examine whether such assemblies can work under cellular crude conditions. It is regarded that these challenges contribute to expand intriguing possibilities of supramolecular chemistry in live cells. Rao and colleagues demonstrated that a designer supramolecular assembly of small molecules could occur in living cells by the bio-orthogonal condensation reaction between 1,2-aminothiol and 2-cyanobenzothiazole (Figure 15 a).[41] The reaction generates macrocyclic compounds self-assembling in living cells, which was observed by CLSM (Figure 15 b). This supramolecular probe was successfully applied to imaging of the proteolytic activity of furin. Furthermore, this group suc-
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Figure 15. (a) Proposed mechanism of condensation of monomers in cells and chemical structures of compounds 29 and 30. (b) CLSM images of HeLa cells incubated with 200 mm of monomer 29 (upper) and 100 mm of control monomer 30 (lower). Left: images of the condensation product, Right: the merged images with nuclear staining with DAPI. Scale bars: 20 mm. (c) Proposed caspase-3/7 and reduction-controlled conversion of 31 into 32 through the bioorthogonal intramolecular cyclization reaction, followed by assembly into nanoaggregates in situ. (d) The proposed mechanism of self-assembly of 31 in vivo. 31 extravasates into tumor tissue. In live tumor tissue that does not respond to applied chemotherapy, the pro-caspase-3 is inactive and the DEVD capping peptide remains intact. In apoptotic tumor tissue, pro-caspase-3 is converted into active caspase-3, and 31 can enter cells. After DEVD cleavage by active caspase-3 and disulfide reduction, 31 undergoes macrocyclization and in situ nanoaggregation, which leads to enhanced probe retention and high fluorescence. (e) Fluoresence imaging with 31 of doxorubicin-treated (upper) and saline-treated (lower) tumor-bearing mice. Adapted from ref. [41] and [42]. Copyright 2009 and 2014 Nature Publishing Group. Chem. Asian J. 2015, 10, 2026 – 2038
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Focus Review ceeded in developing a supramolecular imaging system in vivo (Figure 15 c,d,e).[42] The self-assembly is induced by both GSHmediated cleavage of a disulfide bond and elimination of an Asp-Glu-Val-Asp (DEVD) peptide catalyzed by caspase-3/7. Owing to overexpression of caspase-3/7 in apoptotic cells, these supramolecular assemblies form and accumulate in cancer cells in vivo, which allowed for monitoring cancer in mice. Self-assemblies coupled with AIE are also useful for monitoring an enzymatic reaction in cells.[43] Liu and co-workers reported a supramolecular assembly composed of a DEVD peptide and a hydrophobic tetraphenylethene (TPE) unit. In the monomer state, the molecule is nonfluorescent, whereas the resultant molecules cleaved by caspase-3/7 assemble to become emissive. This supramoelcular probe can monitor real-time cell apotosis. Xu and co-workers succeeded in the formation of supramolecular nanofibers catalyzed by an endogenous enzymatic reaction inside live cells (Figure 16).[44, 45, 46] They used the dephosphorylation reaction of 34 by alkaline phosphatase (ALP) to form supramolecular nanofibers (Figure 16 b). The direct ob-
servation by CLSM revealed that the enzymatic reaction and self-assembly occurred in and near the endoplasmic reticulum (ER) (Figure 16 c). The co-incubation with an inhibitor of ALP decreased the number of cells containing supramolecular nanofibers, which clearly showed that the self-assembly is triggered by endogenous ALP. They also reported that a hydrogelator produced by an enzymatic reaction could form nanonets and hydrogels in the pericellular space around HeLa cells.[47] The rapid formation of the hydrogel is able to entrap secretory phosphatases, thereby inducing cell apotosis. This result demonstrates a new strategy for controlling the cell fate by changing the cellular microenvironment and shows that supramolecular assemblies hold potential as new tools for chemical therapy. The preparation of supramolecular assemblies attracted also attention as a new method for improving drug efficacy. Kasai and colleagues demonstrated that nanosized crystals of the SN-38 (anticancer drug) dimer 37 can be produced by a reprecipitation method (Figure 17).[48] The nanocrystals exhibit a good stability in aqueous dispersions and have an improved cell penetrability compared to that of monomer SN-38. The dimers
Figure 16. (a) Synthetic route of hydrogelator 35 forming supramolecular nanofibers in cells. (b) Schematic representation of imaging enzyme-triggered supramolecular assembly in cells. (c) Enzyme-trigged self-assembly inside live cells. CLSM images show the time course of fluorescence emission inside the HeLa cells incubated with 500 or 50 mm of 34. Scale bar: 50 mm for time course panels and 10 mm for the enlarged panels. Adapted from ref. [44]. Chem. Asian J. 2015, 10, 2026 – 2038
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Focus Review tein labeling was performed using a reactive and hydrolyzable tosyl group,[53] the aggregation of labeling reagents can effectively protect the reactive group from the attack of water to suppress the unfavorable decomposition of labeling reagents.[54]
7. Summary and Perspective
Figure 17. (a) Chemical structures of SN-38 and ester-linked SN-38 dimer 37. (b) SEM image of nanocrystals of ester-linked SN-38 dimer. Adapted from ref. [48].
In this Focus Review, we summarized strategies for the construction of supramolecular assemblies responsive to biomolecules and their functions, which clearly indicates their potential as drug-delivery carriers, medical diagnosis kits, and therapeutic tools. In order to make more advances for practical biomaterials, we need to establish a rational molecular design for constructing supramolecular assemblies in cells or in vivo. Most of the supramolecular assemblies have been applied only in vitro, and the application of supramolecular assemblies in cells is still a difficult task. A quantitative analysis of the in-cell behavior of supramolecular assemblies is highly desired. In addition, hybridization of supramolecular assemblies with various materials such as polymers, inorganic materials, and biomole-
are hydrolized in the cell to afford the original SN-38. Such a simple strategy adequately improves the stability and other properties of drugs. In another example, the conjugation of two distinct hydrophilic and hydrophobic drugs gave a nanosized self-assembling prodrug.[49] The stability of the prodrug was found to be improved compared to that of the corresponding monomer drugs. In addition, the nanoparticles showed a good efficiency for accumulation in tumor tissues due to the en- Figure 18. (a) Schematic illustration of on-cell or in-cell reversible protein sensing by self-assembling nanoaggrehanced permeability and reten- gates. (b) Transmembrane-type hCA imaging of A549 cells using self-assembled probe without (upper) or with tion (EPR) effect. The strategy of (lower) inhibitor of hCA. (c) CLSM images of MCF7 cells treated with self-assembled probe without (upper) or with using a drug–drug conjugate to (lower) inhibitor of a target protein. Adapted from ref. [51] and [52]. Copyright 2012 and 2014 American Chemical Society. form nanoaggregation may open a new way for the improvement of chemotherapy.[50] cules is strongly recommended for reinforcement of supraSelf-assembling materials can be used not only for imaging molecular materials and multifunctionalization. apoptosis and improving the potency of drugs but also for constructing cell-based inhibitor assays.[51] Ligand–dye conjugates (Figure 18) spherically assemble, thus resulting in reKeywords: biological applications · biomolecules · hydrogels · duced fluorescence due to the close packing of dyes. The fluostimuli responsiveness · supramolecular assembly rescence recovered when the nanoaggregates collapsed upon recognition of target proteins. The fluorescent turn-on system can be applied as an inhibitor assay in live cells as well as on [1] a) T. Aida, E. W. Meijer, S. I. Stupp, Science 2012, 335, 813 – 817; b) X. Yan, the cell surface (Figure 18 b,c).[52] F. Wang, B. Zheng, F. Huang, Chem. Soc. Rev. 2012, 41, 6042 – 6065; c) E. The supramolecular assembly may bring a positive effect for Busseron, Y. Ruff, E. Moulin, N. Giuseppone, Nanoscale 2013, 5, 7098 – specific protein labeling under live-cell conditions. When pro7140; d) Z. Qi, C. A. Schalley, Acc. Chem. Res. 2014, 47, 2222 – 2233. Chem. Asian J. 2015, 10, 2026 – 2038
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Focus Review [2] a) A. P. Blum, J. K. Kammeyer, A. M. Rush, C. E. Callmann, M. E. Hahn, N. C. Gianneschi, J. Am. Chem. Soc. 2015, 137, 2140 – 2154. [3] a) J.-O. You, D. Almeda, G. J. Ye, D. T. Auguste, J. Biol. Eng. 2010, 4, 15 – 26; b) K. J. Cash, H. A. Clark, Trends Mol. Med. 2010, 16, 584 – 593; c) J. Hu, G. Zhang, S. Liu, Chem. Soc. Rev. 2012, 41, 5933 – 5949. [4] K. Miyata, N. Nishiyama, K. Kataoka, Chem. Soc. Rev. 2012, 41, 2562 – 2574. [5] a) S. Fleming, R. V. Ulijn, Chem. Soc. Rev. 2014, 43, 8150 – 8177; b) C. Ren, J. Zhang, M. Chen, Z. Yang, Chem. Soc. Rev. 2014, 43, 7257 – 7266. [6] a) M. H. Lee, Z. Yang, C. W. Lim, Y. H. Lee, S. Dongbang, C. Kang, J. S. Kim, Chem. Rev. 2013, 113, 5071 – 5109; b) M. A. Cohen Stuart, W. T. S. Huck, J. Genzer, M. Mìller, C. Ober, M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk, M. Urban, F. Winnik, S. Zauscher, I. Luzinov, S. Minko, Nat. Mater. 2010, 9, 101 – 113. [7] C. Ren, Z. Song, W. Zheng, X. Chen, L. Wang, D. Kong, Z. Yang, Chem. Commun. 2011, 47, 1619 – 1621. [8] a) C. Yang, D. Li, Z. Liu, G. Hong, J. Zhang, D. Kong, Z. Yang, J. Phys. Chem. B 2012, 116, 633 – 638; b) Y. Shi, J. Wang, H. Wang, Y. Hu, X. Chen, Z. Yang, PLOS ONE 2014, 9, e106968. [9] R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen, Z. Zhong, J. Controlled Release 2011, 152, 2 – 12. [10] M. Ikeda, T. Tanida, T. Yoshii, I. Hamachi, Adv. Mater. 2011, 23, 2819 – 2822. [11] M. Ikeda, T. Tanida, T. Yoshii, K. Kurotani, S. Onogi, K. Urayama, I. Hamachi, Nat. Chem. 2014, 6, 511 – 512. [12] X. Miao, W. Cao, W. Zheng, J. Wang, X. Zhang, J. Gao, C. Yang, D. Kong, H. Xu, L. Wang, Z. Yang, Angew. Chem. Int. Ed. 2013, 52, 7781 – 7785; Angew. Chem. 2013, 125, 7935 – 7939. [13] Y. Zhang, H. Gu, Z. Yang, B. Xu, J. Am. Chem. Soc. 2003, 125, 13680 – 13681. [14] Z. Qiu, H. Yu, J. Li, Y. Wang, Y. Zhang, Chem. Commun. 2009, 3342 – 3344. [15] C. M. Micklitsch, P. J. Knerr, M. C. Branco, R. Nagarkar, D. J. Pochan, J. P. Schneider, Angew. Chem. Int. Ed. 2011, 50, 1577 – 1579; Angew. Chem. 2011, 123, 1615 – 1617. [16] H. Komatsu, S. Matsumoto, S. Tamaru, K. Kaneko, M. Ikeda, I. Hamachi, J. Am. Chem. Soc. 2009, 131, 5580 – 5585. [17] Y.-X. Wang, D.-S. Guo, Y. Cao, Y. Liu, RSC Adv. 2013, 3, 8058 – 8063. [18] J. Liu, M. Morikawa, N. Kimizuka, J. Am. Chem. Soc. 2011, 133, 17370 – 17374. [19] S. Bhuniya, B. H. Kim, Chem. Commun. 2006, 1842 – 1844. [20] M. Kumar, P. Brocorens, C. Tonnel¦, D. Beljonne, M. Surin, S. J. George, Nat. Commun. 2014, 5, 5793. [21] M. Ikeda, K. Fukuda, T. Tanida, T. Yoshii, I. Hamachi, Chem. Commun. 2012, 48, 2716 – 2718. [22] S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato, I. Hamachi, Nat. Mater. 2004, 3, 58 – 64. [23] A. Wada, S. Tamaru, M. Ikeda, I. Hamachi, J. Am. Chem. Soc. 2009, 131, 5321 – 5330. [24] M. Ikeda, T. Yoshii, T. Matsui, T. Tanida, H. Komatsu, I. Hamachi, J. Am. Chem. Soc. 2011, 133, 1670 – 1673. [25] Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam, B. Xu, Adv. Mater. 2004, 16, 1440 – 1444. [26] a) Z. Yang, G. Liang, B. Xu, Acc. Chem. Res. 2008, 41, 315 – 326; b) Z. Yang, B. Xu, Adv. Mater. 2006, 18, 3043 – 3046; c) Z. Yang, G. Liang, L. Wang, B. Xu, J. Am. Chem. Soc. 2006, 128, 3038 – 3043; d) Z. Yang, P.-L. Ho, G. Liang, K. H. Chow, Q. Wang, Y. Cao, Z. Guo, B. Xu, J. Am. Chem. Soc. 2007, 129, 266 – 267; e) Z. Yang, G. Liang, M. Ma, Y. Gao, B. Xu, Small 2007, 3, 558 – 562; f) S. Toledano, R. J. Williams, V. Jayawarna, R. V. Ulijn, J. Am. Chem. Soc. 2006, 128, 1070 – 1071.
Chem. Asian J. 2015, 10, 2026 – 2038
www.chemasianj.org
[27] Y. Gao, Y. Kuang, Z.-F. Guo, I. J. Krauss, B. Xu, J. Am. Chem. Soc. 2009, 131, 13576 – 13577. [28] L. Jiang, Y. Yan, M. Drechsler, J. Huang, Chem. Commun. 2012, 48, 7347 – 7349. [29] D. Guo, K. Wang, Y. Wang, Y. Liu, J. Am. Chem. Soc. 2012, 134, 10244 – 10250. [30] D. Guo, T. Zhang, Y. Wang, Y. Liu, Chem. Commun. 2013, 49, 6779 – 6781. [31] J. Boekhoven, M. Koot, T. A. Wezendonk, R. Eelkema, J. H. van Esch, J. Am. Chem. Soc. 2012, 134, 12908 – 12911. [32] K. Matsuo, R. Kamada, K. Mizusawa, H. Imai, Y. Takayama, M. Narazaki, T. Matsuda, Y. Takaoka, I. Hamachi, Chem. Eur. J. 2013, 19, 12875 – 12883. [33] Y. Takaoka, Y. Fukuyama, K. Matsuo, I. Hamachi, Chem. Lett. 2013, 42, 1426 – 1428. [34] M. Wang, X. Gu, G. Zhang, D. Zhang, D. Zhu, Anal. Chem. 2009, 81, 4444 – 4449. [35] X. Zhang, X. Chu, L. Wang, H. Wang, G. Liang, J. Zhang, J. Long, Z. Yang, Angew. Chem. Int. Ed. 2012, 51, 4388 – 4392; Angew. Chem. 2012, 124, 4464 – 4468. [36] Y. Takaoka, T. Sakamoto, S. Tsukiji, M. Narazaki, T. Matsuda, H. Tochio, M. Shirakawa, I. Hamachi, Nat. Chem. 2009, 1, 557 – 561. [37] K. Mizusawa, Y. Ishida, Y. Takaoka, M. Miyagawa, S. Tsukiji, I. Hamachi, J. Am. Chem. Soc. 2010, 132, 7291 – 7293. [38] Y. Takaoka, K. Kiminami, K. Mizusawa, K. Matsuo, M. Narazaki, T. Matsuda, I. Hamachi, J. Am. Chem. Soc. 2011, 133, 11725 – 11731. [39] P. Anees, S. Sreejith, A. Ajayaghosh, J. Am. Chem. Soc. 2014, 136, 13233 – 13239. [40] J. Guo, J. Zhuang, F. Wang, K. R. Raghupathi, S. Thayumanavan, J. Am. Chem. Soc. 2014, 136, 2220 – 2223. [41] G. Liang, H. Ren, J. Rao, Nat. Chem. 2010, 2, 54 – 60. [42] D. Ye, A. J. Shuhendler, L. Cui, L. Tong, S. S. Tee, G. Tikhomirov, D. W. Felsher, J. Rao, Nat. Chem. 2014, 6, 519 – 526. [43] H. Shi, R. T. K. Kwok, J. Liu, B. Xing, B. Z. Tang, B. Liu, J. Am. Chem. Soc. 2012, 134, 17972 – 17981. [44] Y. Gao, J. Shi, D. Yuan, B. Xu, Nat. Commun. 2012, 3, 1033. [45] J. Li, Y. Gao, Y. Kuang, J. Shi, X. Du, J. Zhou, H. Wang, Z. Yang, B. Xu, J. Am. Chem. Soc. 2013, 135, 9907 – 9914. [46] Y. Gao, C. Berciu, Y. Kuang, J. Shi, D. Nicastro, B. Xu, ACS Nano 2013, 7, 9055 – 9063. [47] Y. Kuang, J. F. Shi, J. Li, D. Yuan, K. A. Alberti, Q. Xu, B. Xu, Angew. Chem. Int. Ed. 2014, 53, 8104 – 8107; Angew. Chem. Int. Ed. 2014, 53, 8104 – 8107; Angew. Chem. 2014, 126, 8242 – 8245. [48] H. Kasai, T. Murakami, Y. Ikuta, Y. Koseki, K. Baba, H. Oikawa, H. Nakanishi, M. Okada, M. Shoji, M. Ueda, H. Imahori, M. Hashida, Angew. Chem. Int. Ed. 2012, 51, 10315 – 10318; Angew. Chem. 2012, 124, 10461 – 10464. [49] P. Huang, D. Wang, Y. Su, W. Huang, Y. Zhou, D. Cui, X. Zhu, D. Yan, J. Am. Chem. Soc. 2014, 136, 11748 – 11756. [50] S. C. Owen, A. K. Doak, P. Wassam, M. S. Shoichet, B. K. Shoichet, ACS Chem. Biol. 2012, 7, 1429 – 1435. [51] K. Mizusawa, Y. Takaoka, I. Hamachi, J. Am. Chem. Soc. 2012, 134, 13386 – 13395. [52] T. Yoshii, K. Mizusawa, Y. Takaoka, I. Hamachi, J. Am. Chem. Soc. 2014, 136, 16635 – 16642. [53] S. Tsukiji, M. Miyagawa, Y. Takaoka, T. Tamura, I. Hamachi, Nat. Chem. Biol. 2009, 5, 341 – 343. [54] Y. Takaoka, Y. Sun, S. Tsukiji, I. Hamachi, Chem. Sci. 2011, 2, 511 – 520. Manuscript received: June 1, 2015 Accepted Article published: July 7, 2015 Final Article published: July 30, 2015
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Focus Review
Supramolecular Chemistry
Supramolecular Assemblies Responsive to Biomolecules toward Biological Applications Hajime Shigemitsu[a] and Itaru Hamachi*[a, b]
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Focus Review Abstract: Stimuli-responsive supramolecular assemblies consisting of small molecules are attractive functional materials for biological applications such as drug delivery, medical diagnosis, enzyme immobilization, and tissue engineering. By use of their dynamic and reversible properties, many supramolecular assemblies responsive to a variety of biomolecules have been designed and synthesized. This review focuses on
1. Introduction Much attention has been paid to stimuli-responsive supramolecular assemblies as one of the smart materials in various fields.[1] Among them, supramolecular assemblies responsive to biomolecules are promising as drug-delivery carriers, reagents for detection and imaging of biomolecules, and others.[2] Albeit being in an early stage, a myriad of attempts toward biomedical application using supramolecular materials have been made in recent years. To date, nano-sized biomaterials made of organic polymers and inorganic nanoparticles have been mainly studied because their mechanical toughness and stability are crucial for biological applications.[3] In particular, drug- or gene-delivery vehicles composed of polymers have been remarkably improved in their functions in the last decade.[4] On the other hand, supramolecular assemblies consisting of small molecules have not been well developed in this research direction yet. Neither guidelines for their molecular design nor methods for controlling their aggregation morphologies suitable for biological application have been established. However, the designability of stimuli-responsiveness, reversibility, homogeneity, and finely tunable properties are intriguing in supramolecular assemblies composed of small molecules. With the aim of a rational design of supramolecular assemblies as unique biomaterials, it is apparent that fundamental understanding of the assembly behavior in water and, more favorably, under crude conditions is keenly needed. One of the important features of supramolecular assemblies represents stimuli-responsiveness, which is a key for producing intelligent biomaterials. Installing a response to biomolecule into supramolecular materials would allow for various applications such as drug delivery, medical diagnosis, and imaging and monitoring of specific biomolecules. So far, many researchers have employed supramolecular assemblies composed of peptides and/or amphiphilic lipid derivatives and sought to [a] Dr. H. Shigemitsu, Prof. Dr. I. Hamachi Department of Synthetic Chemistry and Biological Chemistry Graduated School of Engineering Kyoto University Katsura, Kyoto 615-8510 (Japan) E-mail: [email protected] [b] Prof. Dr. I. Hamachi Core Research for Evolutional Science and Technology (CREST) Japan Science and Technology Agency (JST) 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075 (Japan) Chem. Asian J. 2015, 10, 2026 – 2038
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promising strategies for the construction of such dynamic supramolecular assemblies and their functions. While studies of biomolecule-responsive supramolecular assemblies have mainly been performed in vitro, it has recently been demonstrated that some of them can work in live cells. Supramolecular assemblies now open up new avenues in chemical biology and biofunctional materials.
reveal the correlations between the molecular structures and their aggregation/response behaviors.[5] Further progress in unveiling these complicated relationships should lead to the rational design of stimuli-responsive supramolecular assemblies. In addition, intensive studies of dynamic supramolecular assemblies in multi-component systems would contribute not only to creating novel biomaterials but also to shedding light on behaviors of synthetic molecules in living cells. In nature, various molecules cooperatively or orthogonally interact and play roles in maintaining homeostasis and adapting to environmental changes. It is considered that simple supramolecular assemblies comprising multiple components should be a good model of such complex systems, which may lead to the synthesis of cell-mimetic supramolecular systems In this Focus Review, we describe the recent progress in biomolecule-responsive supramolecular assemblies consisting of small molecules, particularly by focusing on micelles, vesicles, and nanofibers (hydrogels). Although these supramolecular materials have been mainly studied in vitro so far, a few recent examples clearly showed that supramolecular assemblies can maintain their aggregation structures and function even under cellular conditions, implying that supramolecular assemblies exhibit much potential for biological applications in cells or in vivo. We start this review with examples of supramolecular assemblies that are responsive to small biomolecules through chemical reactions in vitro. Next, supramolecular assemblies responsive to small-molecule recognition, enzymatic reactions; and protein recognition are sequentially described. Finally, successful applications of several supramolecular assemblies in living cells are summarized.
2. Supramolecular Assemblies Responsive to Small Biomolecules via Chemical Reaction In the first section, we introduce supramolecular assemblies responding to small biomolecules through chemical reactions. Most of such assemblies reported so far are categorized into supramolecular gels. Such gels may be potentially applied in the rapid and convenient diagnosis of biomarkers, since the gel–sol transition is easily detectable by the naked eye. In addition, the controlled drug release may be another attractive application due to the possibility of encapsulating various molecules in the gels. In living cells, there are many small molecules such as various cations and anions, reactive oxygen species (ROS), gluta-
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Focus Review thione (GSH), adenosine triphosphate (ATP), sugars, lipids, and so on. Such small molecules play important roles to maintain homeostasis of living systems. For instance, GSH, a simple tripeptide, is known to control the reductive environment in live cells. ATP is a key energy source for various enzymatic reactions and mechanical motions of proteins. Utilization of such molecules as biological stimuli is expected as a rational strategy for designing bio-responsive materials. Although many synthetic polymers responsive to biomolecules have been reported,[6] supramolecular assemblies exhibiting such functions have been limited. Yang and co-workers showed the first example of GSH-triggered hydrogelation of an oligopeptide scaffold containing a disulfide bond (Figure 1).[7] The designer oligopeptide Nap-
struct supramolecular gels responsive to small molecules via chemical reactions was proposed, which relied on tethering appropriate chemically reactive groups to the N-terminus of short peptides (Figure 2 a).[10] For example, modification of an oligo-phenylalanine peptide with phenylboronic acid 3 at the N-terminus successfully afforded H2O2-responsive hydrogels (Figure 2 b). The appended phenylboronic acid reacts with H2O2 and the subsequently induced 1,6-elimination reaction destroys the molecular structure of the hydrogelator to cause the gel–sol transition. In the cases of nitrobenzene- and hydroxycoumarin-modified peptides (4 and 5), the gel–sol transitions were triggered by reductive reagents (e.g., Na2S2O4, NADH) and photo-irradiation, respectively (Figure 2 b). The Hamachi group constructed supramolecular hydrogels encapsulating several enzymes as new biomarker-responsive gels.[11] The hybrid gels composed of a H2O2-responsive hydrogelator and an oxidase which produces H2O2 as a by-product during substrate oxidation result in gel–sol transition in the presence of a biomarker (i.e., a substrate of an oxidase). This system is applicable for various oxidases and thus provides a new method to expand the responsiveness and possibility as a smart supramolecular gel for easy diagnosis. Yang and colleagues achieved a reversible gel–sol transition system by a redox reaction inspired by natural redox systems (e.g., NADH/NAD) (Figure 3).[12] The peptide containing a selenium atom (7) assembles fibrously and forms supramolecular hydrogels. Upon oxidation by H2O2, the peptide 7 changes to 6 and the self-assembled fibers convert into micelles (Figure 3 b– d). The resultant selenoxide compound 6 could be reduced by
Figure 1. (a) Molecular structures of oligopeptides 1 and 2. (b) Optical images showing hydrogelation after addition of GSH. (c,d) TEM images of self-assembled structures of (c) 2 and (d) 1. Adapted from Ref. [7] with permission from The Royal Society of Chemistry.
Itaru Hamachi obtained PhD 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 of 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.
GFFYE-CS-EERGD 2 is composed of three functional parts (Figure 1 a). The Nap-GFFYE part 1 constructs supramolecular fibers and gelates water, and the EERGD part guarantees a sufficient solubility in water. The disulfide bond (CS unit) connects the two peptide parts, which can be cleaved by GSH to give Nap-GFFYE and hydrogels (Figure 1 b). TEM observations of self-assemblied structures of 1 and 2 show a fibrious and an amorphous structure, respectively (Figure 1 c,d). The GSH-inductive hydrogel does not need any heating operation for gelation, which is favorable for biological applications. The group confirmed the biocompatibility of the gels,[8] suggesting a potential for regenerative medicine and cancer therapy. In the intracellular space, high levels of GSH maintain a reductive environment; by contast, GSH is present at low levels in the extracellular matrix. Therefore, utilizing GSH is a rational development for supramolecular assemblies working only inside living cells.[9] Gel–sol transitions, but not sol–gel transitions, by small biomolecules have also been reported. A rational design to conChem. Asian J. 2015, 10, 2026 – 2038
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Hajime Shigemitsu received his PhD 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 material chemistry, supramolecular chemistry and chemical biology.
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Figure 2. (a) Scheme showing the self-assembly process of peptide-based hydrogelators to form supramolecular hydrogels and their stimuli-responsive gel– sol transition. (b) Stimuli-responsive degradation mechanisms of 3, 4, and 5. Adapted from ref. [10].
Figure 3. (a) Chemical structures of selenium-containing peptides (6 and 7) and optical images of the solution and the gel. (b) Schematic representation of the redox-triggered transformation between micelles of 6 and nanofibers of 7. (c,d) TEM images of the micelles of 6 (c) and nanofibers of 7 (d). Adapted from ref. [12].
vitamin C to restore the original peptide structure and recover the hydrogel state again. Interestingly, the rather small change between selenide and selenoxide critically affects the assembling behavior so as to successfully give the reversible gel–sol transition by biological stimuli.
3. Supramolecular Assemblies Responsive to Molecular Recognition Owing to the relatively weak non-covalent interactions between components of supramolecular assemblies, it is expected that they easily change their morphology and optical property by interacting with guest molecules. Since molecular recognition does not destroy molecular structures unlike chemical reactions, the response relying on molecular recognition may afford reversibly responsive biomaterials. In this section, some of the pioneering works are discussed. Chem. Asian J. 2015, 10, 2026 – 2038
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3.1. Supramolecular Assembly or Disassembly Triggered by Molecular Recognition In 2003, Xu and colleagues reported stimuli-responsive supramolecular hydrogels using ligand–receptor interaction.[13] They exploited a simple hydrogelator 8 and a strong interaction between the d-Ala-d-Ala peptide sequence and vancomycin,[14] a well-known antibiotic, to induce gel–sol transition (Figure 4). This is the first achievement with regard to gel–sol transition triggered by molecular recognition. Schneider and co-workers reported zinc-triggered hydrogelation of self-assembling b-hairpin peptide 9 (Figure 5).[15] Insertion of 3-amidoethoxyaminodiacetoxy-2-aminopropionic acid into the b-hairpin peptide gives a hydrogelator showing the formation of Zn2 + -triggered supramolecular nanofibers and hydrogelation (Figure 5 c). Zn2 + plays an important role in inducing folding and the self-assembly of peptide 9 (Figure 5 a). The control peptide 10 that lacks the chelator part does not display such Zn2 + -responsiveness. Zinc is a crucial metal for life, and
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Focus Review 3.2. Molecular Recognition without Altering the Assembly and the Optical Sensing of Small Biomolecules
Figure 4. (a) Molecular structure of hydrogelator 8 and (A) its hydrogel. The phase transitions of the hydrogel induced by the addition of (B) 0.01, (C) 0.1, and (D) 1.0 equiv of solid vancomycin into the hydrogel. (b) Schematic representation of ligand–receptor interactions between vancomycin and the gelator. The interaction induces the gel–sol phase transition. Adapted from ref. [13]. Copyright 2003 American Chemical Society.
Figure 5. (a) Proposed mechanism of metal-triggered folding and self-assembly of 9. (b) Primary sequences of gelator 9 showing Zn2 + -responsiveness and the control peptide 10. (c) Optical images of the gel and solution of gelator 9. Adapted from ref. [15].
Zn2 + ions promote matrix metalloproteinase debridement and keratinocyte migration to accelerate wound repair. The amount of Zn2 + present in the hydrogel network is controlled by the amount of self-assembling b-hairpin peptide. Zn2 + would be deliverable to a wound bed using such a gel system. In another example of metal-ion-induced hydrogelation, a lipid-like molecule having a phosphate group shows gelation by addition of Ca2 + ions.[16] Observation by confocal laser scanning microscopy (CLSM) revealed that Ca2 + ions bridge nanofibers and enhance the strength of the fibril networks. Host–guest interactions are also employed for the construction of supramolecular vesicles. Liu and co-workers reported that complexation of ATP with an amphiphilic calixarene 11 produces hollow spherical nanoparticles (Figure 6).[17] The critical micellar concentration of the calixarene 11 largely decreased by ATP binding due to charge neutralization. Interestingly, the hydrolysis of encapsulated ATP by phosphatase destroys the self-assembled vesicles. The assembly and disassembly of such vesicles, depending on ATP encapsulation, took place reversibly. Chem. Asian J. 2015, 10, 2026 – 2038
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In some supramolecular assemblies, only their optical properties were altered upon binding of guest molecules without any morphological changes. Kimizuka and co-workers utilized the vesicle surface for luminescent ATP sensing (Figure 7).[18] The vesicle composed of amphiphilic TbIII complex 12-TbIII senses ATP and ADP by exchange of ligands coordinated to TbIII from H2O to phosphate groups. The ligand exchange causes a sigmoidal increase in luminescent intensity due to the difference in the energy transfer from ligands to TbIII. The high density of cationic TbIII complex on the surface allowed the sensitive detection of low concentrations of ATP. In another example, it was reported that sugar-based hydrogels can be used to detect low concentrations of insulin through changes in the fluorescent intensity, without changes in their fibrous morphology.[19] Chiroptical properties of selfassembly induced by ATP derivatives were successfully used to monitor enzymatic ATP hydroly-
Figure 6. Schematic illustration of the self-assembly of 11 with ATP and its phosphatase response. Adapted from Ref. [17] with permission from The Royal Society of Chemistry.
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Focus Review materials, and enzymes can be concurrently embedded in supramolecular gels while retaining their orginal activities and functions. For instance, supramolecular gels hybridized with fluorescent dyes containing inorganic materials (Montmorillonite (MMT) or mesoporous silica particles (MCM41)) can work as a fluorescent sensor for biologically important polyamines or polyanions (Figure 9).[23, 24] These results also revealed the new possibility of generating supramolecular gels consisting of multiple components.
4. Supramolecular Assemblies Responsive to Enzymatic Reactions Figure 7. Schematic representation for self-assembly of amphiphilic 12-TbIII and binding of ATP molecules. Adapted from ref. [18]. Copyright 2011 American Chemical Society.
sis (Figure 8).[20] The supramolecular polymer consisting of a naphthalene bisimide derivative equipped with dipicolylamine Zn2 + complex 13 constructs a P-type supramolecular helix upon ATP binding (Figure 8 a). On the other hand, ADP or AMP complexation induced an M-type helix. This chiral inversion phenomenon is available for monitoring the enzymatic activity (Figure 8 b). Fluidic circumstances formed by supramolecular gels provide a unique matrix for sensing various small biomolecules.[21, 22] Hamachi and co-workers demonstrated that various functional materials such as dyes, layered and porous inorganic
Some particular enzymes are overexpressed in disease-related cells and tissues, and thus supramolecular assemblies responsive to such enzymatic reactions could lead to chemotherapeutic and diagnostic tools. Given that enzymes catalyze the formation or cleavage of chemical bonds with high selectivity, the incorporation of a module susceptible to an enzymatic reaction into a component of supramolecular assemblies is a promising strategy for rational design to produce a supramolecular assembly responsive to a specific enzyme. 4.1. Gelation and Gel–Sol Transition Triggered by Enzymatic Reactions
Figure 8. (a) Molecular structure of NDPA 13 along with a pictorial representation of adenosine phosphates (APs) inducing a helical supramolecular organization. (b) Schematic illustration of the dynamic helix reversals upon enzyme (CIAP) action on the NDPA-bound ATP molecules and their respective real-time chiroptical readout. Adapted from ref. [20]. Copyright 2014 Nature Publishing Group. Chem. Asian J. 2015, 10, 2026 – 2038
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Xu and colleagues reported supramolecular hydrogel formation triggered by an enzymatic reaction in 2004 (Figure 10).[25] Dephosphorylation of a simple amino acid derivative by a phosphatase resulted in a hydrogelator and subsequent formation of hydrogels. Conversion of an anionic group into a neutral group can effectively change the balance of the hydrophobicity and hydrophilicity to yield a hydrogelator. After this report, various hydrogelators generated by many types of enzymatic reactions (kinase, lactamase, peptidase, and esterase) have been developed.[26]
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Focus Review Hydrogelation by enzymatic reactions is applicable for improvement of drug dispersity. The group of Xu synthesized a hydrogelator precursor containing taxol, which is a potent anticancer drug in spite of its poor solubility and dispersibility in water.[27] The enzyme-triggered hydrogelation yielded supramolecular hydrogels containing taxol without loss of its potency. 4.2. Multicomponent Systems of Supramolecular Assemblies Responsive to Enzymatic Reactions
Figure 9. Hybrid materials of supramolecular gels and inorganic materials (a: MCM41, b: MMT). (a) Chemical structures of compounds used in this work (Suc-8S, IP6, heparin, and chondroitin sulfate are the biomolecules sensed in this system), and construction and mechanism operating in the fluorescent dye-encapsulated MCM/enzyme/ supramolecular hydrogel hybrid sensory system. (b) Chemical structures of compounds used in this work (spermine and spermidine are the anionic biomolecules detected by this system), and construction and the mechanism of action of the fluorescence dye (G-coum) adsorbed MMT/supramolecular hydrogel 18 hybrid sensory system for polyamines. Adapted from ref. [23] and [24]. Copyright 2009 and 2011 American Chemical Society. Chem. Asian J. 2015, 10, 2026 – 2038
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Multicomponent systems of supramolecular assemblies are also intriguing for the design of enzyme-responsive supramolecular assemblies. Huang and colleagues demonstrated enzymetriggered supramolecular assemblies by a host–guest system between cyclodextrin (CD) and surfactants.[28] While these host– guest complexes do not aggregate in water, amylase decomposes CDs to release the corresponding surfactants that form supramolecular assemblies. Liu and co-workers reported enzyme-triggered disassembly of vesicles consisting of host–guest complexes of calixarene derivatives and myristoylcholine.[29] The embedded drugs can be released when these vesicles are destroyed by cholinesterase capable of hydrolyzing myristoylcholine.[30] van Esch and co-workers developed a three-component hydrogel consisting of supramolecular fibers, vesicles, and an enzyme (chymotrypsin).[31] Interestingly, the supramolecular fibers of 21 and the vesicles containing the enzyme orthogonally exist in the hydrogel matrix (Figure 11). The enzyme molecules encapsulated in the vesicles could decompose the gel fibers only when they are re-
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Focus Review metalloproteinase-2 (MMP2) assembled to form nanoparticles (Figure 12 c).[32, 33] In the nanoparticles, the 19F NMR signal is not detected due to the increase in their apparent molecular weight. After collapse of the nanoparticles by MMP2, the 19F NMR signal sharply recovered (FigFigure 10. (a) Chemical structures of the hydrogelator precursor 19 and hydrogelator 20. (b) Optical images of soure 2 b). This turn-on 19F NMR lution of 19 and gel of 20. 19 is hydrolyzed by phosphatase to give 20 and hydrogels. Adapted from ref. [25]. signal-detection system was successfully applied to monitoring the MMP2 activity. Aggregation-induced emission (AIE) is exploited for fluoresleased. The release rate of the enzyme can be tuned by the heating temperature and time, and thus precise control of the cent monitoring of an enzymatic activity. Cationic myristorylgel–sol transition is achieved. choline and anionic tetraphenylethylene (TPE) derivatives interacted with each other in water and form nanoparticles.[34] The resultant nanoparticles show a strong emission due to AIE of 4.3. Optical Sensing and Monitoring of Enzymatic Reactions TPE. After the nanoparticles are destroyed by choline esterases, by Supramolecular Assemblies the emission is reduced, thus enabling monitoring of the cholineesterase activity in a turn-off mode. Turn-on specific detection of enzymatic activities is of fundamental importance in drug discovery and medical diagnosis. Several self-assembling nano-aggregates are applicable to turn-on type imaging of enzymatic activity. Hamachi and co5. Supramolecular Assemblies Responsive to workers reported that amphiphilic molecules 22, 23, and 24 Biopolymers other than Enzymes having a 19F-modified peptide sequence cleavable by matrix There are various biopolymers (e.g., proteins, DNA, RNA, and oligo- or polysaccharides) other than enzymes in cells. Some of them are closely related to various diseases, which makes them important targets to be responded to or to be detected. However, it is much more difficult to design supramolecular assemblies that can respond to such biopolymers, compared to enzymes. This is because these biopolymers simply exhibit
Figure 11. (a) Chemical structure of gelator 21 bearing fluorogenic 6-AQ. (b) Self-assembled multicomponent system: a solution of 21 in DMSO is added to an aqueous solution of the enzyme chymotripsin loaded in liposomes. Heating the gel results in partial liberation of the enzyme into the gel matrix, allowing for release of 6-AQ through enzymatic hydrolysis of the amide bond. Adapted from ref. [31]. Copyright 2012 American Chemical Society. Chem. Asian J. 2015, 10, 2026 – 2038
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Figure 12. (a) Chemical structures of self-assembled probes. (b) Schematic illustration of a substrate-based self-assembling 19F probe for turn-on detection of enzymatic activity. (c) Fluorescent microscopy image of probe 22 (50 mm) loaded with Nile Red. Scale bar: 5 mm. Adapted from ref. [32].
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Figure 13. (a) Chemical structure of Nap-GFFYGGGWRESAI (nongelator) 25 with a possible self-assembly ability. (b–d) Nap-GFFYGGGWRESAI assembles into nanofibers which do not interact in aqueous solution, thus resulting in fiber networks with low density of cross-linking points. (e–g) The addition of rationally designed fusion protein (ULD-TIP-1) enhances the interactions between fibers, thus leading to hydrogelation. Scale bars: 500 nm. Adapted from ref. [35].
a molecular recognition capability, but do not catalyze any chemical reactions. The construction of such supramolecular assemblies requires an elaborate control of non-covalent interactions depending on these biopolymers. Yang and co-workers reported hydrogel formation triggered by protein recognition.[35] They used a fusion protein (ULD-TIP1) as a glue between supramolecular nanofibers of 25 containing an oligopeptide recognized by TIP-1 (Figure 13). ULD-TIP1 is composed of a ubiquitin-like domain (ULD) from the special AT-rich sequence binding protein-1 (SATB-1) and Tax-interaction protein-1 (TIP-1). Since ULD proteins predominantly form a tetramer, ULD-TIP-1 crosslinks the supramolecular nanofibers to afford hydrogels upon TIP-1 interaction with the fibers. The obtained gels showed thixotropic fast gelation properties (Figure 13 e,f,g). It is well known that proteins interact with specific small molecules termed protein ligands. The ligand–protein recognition is selective and thus useful for the development of dynamic biomaterials. In 2009, Hamachi and co-workers found that self-assembled nanoparticles consisting of ligand–probe conjugates can selectively respond to a protein and thus are applicable to protein sensing (Figure 14 ).[36] A molecule containing a protein ligand and fluorine atoms spherically self-assembles and shows no 19F NMR signal due to an increase in the apparent molecular weight. The 19F NMR signal was recovered by disassembly of these nanoparticles upon interaction of a target protein with the ligand part of nanoparticles. This assembly/disassembly principle induced by protein recognition was extended from 19F NMR (MRI) to fluorescent detection. Various ligands and fluorescent dyes such as boron dipyrromethene (BODIPY), fluorescein, and tetramethyl rhodamine (TMR) Chem. Asian J. 2015, 10, 2026 – 2038
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Figure 14. Off/on 19F NMR probes for protein imaging. (a) Strategy used for an off/on switching system of 19F NMR. (b) Chemical structures of the probes with various ligands. Adapted from ref. [36]. Copyright 2009 Nature Publishing Group.
are available for this detection system.[37, 38] This study also highlighted the significance of disassembly of supramolecules, in addition to providing a new sensing strategy for protein detection of proteins. Ajayaghosh and colleagues reported self-assembled nanoparticles composed of near-IR squaraine dyes that specifically reacted with human serum albumin (HSA).[39] The nanoparticles can sense serum albumin protein (SAP) even in the presence of other competing thiol-containing proteins and small molecules. Thayumanavan and co-workers constructed a supramolecular assembly equipped with logic-gate-like responsiveness for enzymatic reaction and protein recognition.[40] In contrast to protein-responsive supramolecules, dynamic supramolecular assemblies responsive to DNA, RNA, and saccharides have been poorly developed to date. These should be explored in the near future.
6. Application of Supramolecular Assemblies in Cells and in Vivo Encouraged by successful examples of supramolecular assemblies responsive to biomolecules in vitro, several groups recently sought to examine whether such assemblies can work under cellular crude conditions. It is regarded that these challenges contribute to expand intriguing possibilities of supramolecular chemistry in live cells. Rao and colleagues demonstrated that a designer supramolecular assembly of small molecules could occur in living cells by the bio-orthogonal condensation reaction between 1,2-aminothiol and 2-cyanobenzothiazole (Figure 15 a).[41] The reaction generates macrocyclic compounds self-assembling in living cells, which was observed by CLSM (Figure 15 b). This supramolecular probe was successfully applied to imaging of the proteolytic activity of furin. Furthermore, this group suc-
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Figure 15. (a) Proposed mechanism of condensation of monomers in cells and chemical structures of compounds 29 and 30. (b) CLSM images of HeLa cells incubated with 200 mm of monomer 29 (upper) and 100 mm of control monomer 30 (lower). Left: images of the condensation product, Right: the merged images with nuclear staining with DAPI. Scale bars: 20 mm. (c) Proposed caspase-3/7 and reduction-controlled conversion of 31 into 32 through the bioorthogonal intramolecular cyclization reaction, followed by assembly into nanoaggregates in situ. (d) The proposed mechanism of self-assembly of 31 in vivo. 31 extravasates into tumor tissue. In live tumor tissue that does not respond to applied chemotherapy, the pro-caspase-3 is inactive and the DEVD capping peptide remains intact. In apoptotic tumor tissue, pro-caspase-3 is converted into active caspase-3, and 31 can enter cells. After DEVD cleavage by active caspase-3 and disulfide reduction, 31 undergoes macrocyclization and in situ nanoaggregation, which leads to enhanced probe retention and high fluorescence. (e) Fluoresence imaging with 31 of doxorubicin-treated (upper) and saline-treated (lower) tumor-bearing mice. Adapted from ref. [41] and [42]. Copyright 2009 and 2014 Nature Publishing Group. Chem. Asian J. 2015, 10, 2026 – 2038
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Focus Review ceeded in developing a supramolecular imaging system in vivo (Figure 15 c,d,e).[42] The self-assembly is induced by both GSHmediated cleavage of a disulfide bond and elimination of an Asp-Glu-Val-Asp (DEVD) peptide catalyzed by caspase-3/7. Owing to overexpression of caspase-3/7 in apoptotic cells, these supramolecular assemblies form and accumulate in cancer cells in vivo, which allowed for monitoring cancer in mice. Self-assemblies coupled with AIE are also useful for monitoring an enzymatic reaction in cells.[43] Liu and co-workers reported a supramolecular assembly composed of a DEVD peptide and a hydrophobic tetraphenylethene (TPE) unit. In the monomer state, the molecule is nonfluorescent, whereas the resultant molecules cleaved by caspase-3/7 assemble to become emissive. This supramoelcular probe can monitor real-time cell apotosis. Xu and co-workers succeeded in the formation of supramolecular nanofibers catalyzed by an endogenous enzymatic reaction inside live cells (Figure 16).[44, 45, 46] They used the dephosphorylation reaction of 34 by alkaline phosphatase (ALP) to form supramolecular nanofibers (Figure 16 b). The direct ob-
servation by CLSM revealed that the enzymatic reaction and self-assembly occurred in and near the endoplasmic reticulum (ER) (Figure 16 c). The co-incubation with an inhibitor of ALP decreased the number of cells containing supramolecular nanofibers, which clearly showed that the self-assembly is triggered by endogenous ALP. They also reported that a hydrogelator produced by an enzymatic reaction could form nanonets and hydrogels in the pericellular space around HeLa cells.[47] The rapid formation of the hydrogel is able to entrap secretory phosphatases, thereby inducing cell apotosis. This result demonstrates a new strategy for controlling the cell fate by changing the cellular microenvironment and shows that supramolecular assemblies hold potential as new tools for chemical therapy. The preparation of supramolecular assemblies attracted also attention as a new method for improving drug efficacy. Kasai and colleagues demonstrated that nanosized crystals of the SN-38 (anticancer drug) dimer 37 can be produced by a reprecipitation method (Figure 17).[48] The nanocrystals exhibit a good stability in aqueous dispersions and have an improved cell penetrability compared to that of monomer SN-38. The dimers
Figure 16. (a) Synthetic route of hydrogelator 35 forming supramolecular nanofibers in cells. (b) Schematic representation of imaging enzyme-triggered supramolecular assembly in cells. (c) Enzyme-trigged self-assembly inside live cells. CLSM images show the time course of fluorescence emission inside the HeLa cells incubated with 500 or 50 mm of 34. Scale bar: 50 mm for time course panels and 10 mm for the enlarged panels. Adapted from ref. [44]. Chem. Asian J. 2015, 10, 2026 – 2038
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Focus Review tein labeling was performed using a reactive and hydrolyzable tosyl group,[53] the aggregation of labeling reagents can effectively protect the reactive group from the attack of water to suppress the unfavorable decomposition of labeling reagents.[54]
7. Summary and Perspective
Figure 17. (a) Chemical structures of SN-38 and ester-linked SN-38 dimer 37. (b) SEM image of nanocrystals of ester-linked SN-38 dimer. Adapted from ref. [48].
In this Focus Review, we summarized strategies for the construction of supramolecular assemblies responsive to biomolecules and their functions, which clearly indicates their potential as drug-delivery carriers, medical diagnosis kits, and therapeutic tools. In order to make more advances for practical biomaterials, we need to establish a rational molecular design for constructing supramolecular assemblies in cells or in vivo. Most of the supramolecular assemblies have been applied only in vitro, and the application of supramolecular assemblies in cells is still a difficult task. A quantitative analysis of the in-cell behavior of supramolecular assemblies is highly desired. In addition, hybridization of supramolecular assemblies with various materials such as polymers, inorganic materials, and biomole-
are hydrolized in the cell to afford the original SN-38. Such a simple strategy adequately improves the stability and other properties of drugs. In another example, the conjugation of two distinct hydrophilic and hydrophobic drugs gave a nanosized self-assembling prodrug.[49] The stability of the prodrug was found to be improved compared to that of the corresponding monomer drugs. In addition, the nanoparticles showed a good efficiency for accumulation in tumor tissues due to the en- Figure 18. (a) Schematic illustration of on-cell or in-cell reversible protein sensing by self-assembling nanoaggrehanced permeability and reten- gates. (b) Transmembrane-type hCA imaging of A549 cells using self-assembled probe without (upper) or with tion (EPR) effect. The strategy of (lower) inhibitor of hCA. (c) CLSM images of MCF7 cells treated with self-assembled probe without (upper) or with using a drug–drug conjugate to (lower) inhibitor of a target protein. Adapted from ref. [51] and [52]. Copyright 2012 and 2014 American Chemical Society. form nanoaggregation may open a new way for the improvement of chemotherapy.[50] cules is strongly recommended for reinforcement of supraSelf-assembling materials can be used not only for imaging molecular materials and multifunctionalization. apoptosis and improving the potency of drugs but also for constructing cell-based inhibitor assays.[51] Ligand–dye conjugates (Figure 18) spherically assemble, thus resulting in reKeywords: biological applications · biomolecules · hydrogels · duced fluorescence due to the close packing of dyes. The fluostimuli responsiveness · supramolecular assembly rescence recovered when the nanoaggregates collapsed upon recognition of target proteins. The fluorescent turn-on system can be applied as an inhibitor assay in live cells as well as on [1] a) T. Aida, E. W. Meijer, S. I. Stupp, Science 2012, 335, 813 – 817; b) X. Yan, the cell surface (Figure 18 b,c).[52] F. Wang, B. Zheng, F. Huang, Chem. Soc. Rev. 2012, 41, 6042 – 6065; c) E. The supramolecular assembly may bring a positive effect for Busseron, Y. Ruff, E. Moulin, N. Giuseppone, Nanoscale 2013, 5, 7098 – specific protein labeling under live-cell conditions. When pro7140; d) Z. Qi, C. A. Schalley, Acc. Chem. Res. 2014, 47, 2222 – 2233. Chem. Asian J. 2015, 10, 2026 – 2038
www.chemasianj.org
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Focus Review [2] a) A. P. Blum, J. K. Kammeyer, A. M. Rush, C. E. Callmann, M. E. Hahn, N. C. Gianneschi, J. Am. Chem. Soc. 2015, 137, 2140 – 2154. [3] a) J.-O. You, D. Almeda, G. J. Ye, D. T. Auguste, J. Biol. Eng. 2010, 4, 15 – 26; b) K. J. Cash, H. A. Clark, Trends Mol. Med. 2010, 16, 584 – 593; c) J. Hu, G. Zhang, S. Liu, Chem. Soc. Rev. 2012, 41, 5933 – 5949. [4] K. Miyata, N. Nishiyama, K. Kataoka, Chem. Soc. Rev. 2012, 41, 2562 – 2574. [5] a) S. Fleming, R. V. Ulijn, Chem. Soc. Rev. 2014, 43, 8150 – 8177; b) C. Ren, J. Zhang, M. Chen, Z. Yang, Chem. Soc. Rev. 2014, 43, 7257 – 7266. [6] a) M. H. Lee, Z. Yang, C. W. Lim, Y. H. Lee, S. Dongbang, C. Kang, J. S. Kim, Chem. Rev. 2013, 113, 5071 – 5109; b) M. A. Cohen Stuart, W. T. S. Huck, J. Genzer, M. Mìller, C. Ober, M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk, M. Urban, F. Winnik, S. Zauscher, I. Luzinov, S. Minko, Nat. Mater. 2010, 9, 101 – 113. [7] C. Ren, Z. Song, W. Zheng, X. Chen, L. Wang, D. Kong, Z. Yang, Chem. Commun. 2011, 47, 1619 – 1621. [8] a) C. Yang, D. Li, Z. Liu, G. Hong, J. Zhang, D. Kong, Z. Yang, J. Phys. Chem. B 2012, 116, 633 – 638; b) Y. Shi, J. Wang, H. Wang, Y. Hu, X. Chen, Z. Yang, PLOS ONE 2014, 9, e106968. [9] R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen, Z. Zhong, J. Controlled Release 2011, 152, 2 – 12. [10] M. Ikeda, T. Tanida, T. Yoshii, I. Hamachi, Adv. Mater. 2011, 23, 2819 – 2822. [11] M. Ikeda, T. Tanida, T. Yoshii, K. Kurotani, S. Onogi, K. Urayama, I. Hamachi, Nat. Chem. 2014, 6, 511 – 512. [12] X. Miao, W. Cao, W. Zheng, J. Wang, X. Zhang, J. Gao, C. Yang, D. Kong, H. Xu, L. Wang, Z. Yang, Angew. Chem. Int. Ed. 2013, 52, 7781 – 7785; Angew. Chem. 2013, 125, 7935 – 7939. [13] Y. Zhang, H. Gu, Z. Yang, B. Xu, J. Am. Chem. Soc. 2003, 125, 13680 – 13681. [14] Z. Qiu, H. Yu, J. Li, Y. Wang, Y. Zhang, Chem. Commun. 2009, 3342 – 3344. [15] C. M. Micklitsch, P. J. Knerr, M. C. Branco, R. Nagarkar, D. J. Pochan, J. P. Schneider, Angew. Chem. Int. Ed. 2011, 50, 1577 – 1579; Angew. Chem. 2011, 123, 1615 – 1617. [16] H. Komatsu, S. Matsumoto, S. Tamaru, K. Kaneko, M. Ikeda, I. Hamachi, J. Am. Chem. Soc. 2009, 131, 5580 – 5585. [17] Y.-X. Wang, D.-S. Guo, Y. Cao, Y. Liu, RSC Adv. 2013, 3, 8058 – 8063. [18] J. Liu, M. Morikawa, N. Kimizuka, J. Am. Chem. Soc. 2011, 133, 17370 – 17374. [19] S. Bhuniya, B. H. Kim, Chem. Commun. 2006, 1842 – 1844. [20] M. Kumar, P. Brocorens, C. Tonnel¦, D. Beljonne, M. Surin, S. J. George, Nat. Commun. 2014, 5, 5793. [21] M. Ikeda, K. Fukuda, T. Tanida, T. Yoshii, I. Hamachi, Chem. Commun. 2012, 48, 2716 – 2718. [22] S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato, I. Hamachi, Nat. Mater. 2004, 3, 58 – 64. [23] A. Wada, S. Tamaru, M. Ikeda, I. Hamachi, J. Am. Chem. Soc. 2009, 131, 5321 – 5330. [24] M. Ikeda, T. Yoshii, T. Matsui, T. Tanida, H. Komatsu, I. Hamachi, J. Am. Chem. Soc. 2011, 133, 1670 – 1673. [25] Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam, B. Xu, Adv. Mater. 2004, 16, 1440 – 1444. [26] a) Z. Yang, G. Liang, B. Xu, Acc. Chem. Res. 2008, 41, 315 – 326; b) Z. Yang, B. Xu, Adv. Mater. 2006, 18, 3043 – 3046; c) Z. Yang, G. Liang, L. Wang, B. Xu, J. Am. Chem. Soc. 2006, 128, 3038 – 3043; d) Z. Yang, P.-L. Ho, G. Liang, K. H. Chow, Q. Wang, Y. Cao, Z. Guo, B. Xu, J. Am. Chem. Soc. 2007, 129, 266 – 267; e) Z. Yang, G. Liang, M. Ma, Y. Gao, B. Xu, Small 2007, 3, 558 – 562; f) S. Toledano, R. J. Williams, V. Jayawarna, R. V. Ulijn, J. Am. Chem. Soc. 2006, 128, 1070 – 1071.
Chem. Asian J. 2015, 10, 2026 – 2038
www.chemasianj.org
[27] Y. Gao, Y. Kuang, Z.-F. Guo, I. J. Krauss, B. Xu, J. Am. Chem. Soc. 2009, 131, 13576 – 13577. [28] L. Jiang, Y. Yan, M. Drechsler, J. Huang, Chem. Commun. 2012, 48, 7347 – 7349. [29] D. Guo, K. Wang, Y. Wang, Y. Liu, J. Am. Chem. Soc. 2012, 134, 10244 – 10250. [30] D. Guo, T. Zhang, Y. Wang, Y. Liu, Chem. Commun. 2013, 49, 6779 – 6781. [31] J. Boekhoven, M. Koot, T. A. Wezendonk, R. Eelkema, J. H. van Esch, J. Am. Chem. Soc. 2012, 134, 12908 – 12911. [32] K. Matsuo, R. Kamada, K. Mizusawa, H. Imai, Y. Takayama, M. Narazaki, T. Matsuda, Y. Takaoka, I. Hamachi, Chem. Eur. J. 2013, 19, 12875 – 12883. [33] Y. Takaoka, Y. Fukuyama, K. Matsuo, I. Hamachi, Chem. Lett. 2013, 42, 1426 – 1428. [34] M. Wang, X. Gu, G. Zhang, D. Zhang, D. Zhu, Anal. Chem. 2009, 81, 4444 – 4449. [35] X. Zhang, X. Chu, L. Wang, H. Wang, G. Liang, J. Zhang, J. Long, Z. Yang, Angew. Chem. Int. Ed. 2012, 51, 4388 – 4392; Angew. Chem. 2012, 124, 4464 – 4468. [36] Y. Takaoka, T. Sakamoto, S. Tsukiji, M. Narazaki, T. Matsuda, H. Tochio, M. Shirakawa, I. Hamachi, Nat. Chem. 2009, 1, 557 – 561. [37] K. Mizusawa, Y. Ishida, Y. Takaoka, M. Miyagawa, S. Tsukiji, I. Hamachi, J. Am. Chem. Soc. 2010, 132, 7291 – 7293. [38] Y. Takaoka, K. Kiminami, K. Mizusawa, K. Matsuo, M. Narazaki, T. Matsuda, I. Hamachi, J. Am. Chem. Soc. 2011, 133, 11725 – 11731. [39] P. Anees, S. Sreejith, A. Ajayaghosh, J. Am. Chem. Soc. 2014, 136, 13233 – 13239. [40] J. Guo, J. Zhuang, F. Wang, K. R. Raghupathi, S. Thayumanavan, J. Am. Chem. Soc. 2014, 136, 2220 – 2223. [41] G. Liang, H. Ren, J. Rao, Nat. Chem. 2010, 2, 54 – 60. [42] D. Ye, A. J. Shuhendler, L. Cui, L. Tong, S. S. Tee, G. Tikhomirov, D. W. Felsher, J. Rao, Nat. Chem. 2014, 6, 519 – 526. [43] H. Shi, R. T. K. Kwok, J. Liu, B. Xing, B. Z. Tang, B. Liu, J. Am. Chem. Soc. 2012, 134, 17972 – 17981. [44] Y. Gao, J. Shi, D. Yuan, B. Xu, Nat. Commun. 2012, 3, 1033. [45] J. Li, Y. Gao, Y. Kuang, J. Shi, X. Du, J. Zhou, H. Wang, Z. Yang, B. Xu, J. Am. Chem. Soc. 2013, 135, 9907 – 9914. [46] Y. Gao, C. Berciu, Y. Kuang, J. Shi, D. Nicastro, B. Xu, ACS Nano 2013, 7, 9055 – 9063. [47] Y. Kuang, J. F. Shi, J. Li, D. Yuan, K. A. Alberti, Q. Xu, B. Xu, Angew. Chem. Int. Ed. 2014, 53, 8104 – 8107; Angew. Chem. Int. Ed. 2014, 53, 8104 – 8107; Angew. Chem. 2014, 126, 8242 – 8245. [48] H. Kasai, T. Murakami, Y. Ikuta, Y. Koseki, K. Baba, H. Oikawa, H. Nakanishi, M. Okada, M. Shoji, M. Ueda, H. Imahori, M. Hashida, Angew. Chem. Int. Ed. 2012, 51, 10315 – 10318; Angew. Chem. 2012, 124, 10461 – 10464. [49] P. Huang, D. Wang, Y. Su, W. Huang, Y. Zhou, D. Cui, X. Zhu, D. Yan, J. Am. Chem. Soc. 2014, 136, 11748 – 11756. [50] S. C. Owen, A. K. Doak, P. Wassam, M. S. Shoichet, B. K. Shoichet, ACS Chem. Biol. 2012, 7, 1429 – 1435. [51] K. Mizusawa, Y. Takaoka, I. Hamachi, J. Am. Chem. Soc. 2012, 134, 13386 – 13395. [52] T. Yoshii, K. Mizusawa, Y. Takaoka, I. Hamachi, J. Am. Chem. Soc. 2014, 136, 16635 – 16642. [53] S. Tsukiji, M. Miyagawa, Y. Takaoka, T. Tamura, I. Hamachi, Nat. Chem. Biol. 2009, 5, 341 – 343. [54] Y. Takaoka, Y. Sun, S. Tsukiji, I. Hamachi, Chem. Sci. 2011, 2, 511 – 520. Manuscript received: June 1, 2015 Accepted Article published: July 7, 2015 Final Article published: July 30, 2015
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