A Combination of Bio-Orthogonal Supramolecular Clicking and Proximity Chemical Tagging as a Supramolecular Tool for Discovery of Putative Proteins Associated with Laminopathic Disease.

Protein mutations alter protein-protein interactions that can lead to a number of illnesses. Mutations in lamin A (LMNA) have been reported to cause laminopathies. However, the proteins associated with the LMNA mutation have mostly remained unexplored. Herein, a new chemical tool for proximal proteomics is reported, developed by a combination of proximity chemical tagging and a bio-orthogonal supramolecular latching based on cucurbit[7]uril (CB[7])-based host-guest interactions. As this host-guest interaction acts as a noncovalent clickable motif that can be unclicked on-demand, this new chemical tool is exploited for reliable detection of the proximal proteins of LMNA and its mutant that causes laminopathic dilated cardiomyopathy (DCM). Most importantly, a comparison study reveals, for the first time, mutant-dependent alteration in LMNA proteomic environments, which allows to identify putative laminopathic DCM-linked proteins including FOXJ3 and CELF2. This study demonstrates the feasibility of this chemical tool for reliable proximal proteomics, and its immense potential as a new research platform for discovering biomarkers associated with protein mutation-linked diseases.

Lipid-Oriented Live-Cell Distinction of B and T Lymphocytes.

The identification of each cell type is essential for understanding multicellular communities. Antibodies set as biomarkers have been the main toolbox for cell-type recognition, and chemical probes are emerging surrogates. Herein we report the first small-molecule probe, CDgB, to discriminate B lymphocytes from T lymphocytes, which was previously impossible without the help of antibodies. Through the study of the origin of cell specificity, we discovered an unexpected novel mechanism of membrane-oriented live-cell distinction. B cells maintain higher flexibility in their cell membrane than T cells and accumulate the lipid-like probe CDgB more preferably. Because B and T cells share common ancestors, we tracked the cell membrane changes of the progenitor cells and disclosed the dynamic reorganization of the membrane properties over the lymphocyte differentiation progress. This study casts an orthogonal strategy for the small-molecule cell identifier and enriches the toolbox for live-cell distinction from complex cell communities.

Purification of protein therapeutics via high-affinity supramolecular host-guest interactions.

Efficient purification is crucial to providing large quantities of recombinant therapeutic proteins, such as monoclonal antibodies and cytokines. However, affinity techniques for manufacturing protein therapeutics that use biomolecule-conjugated agarose beads that harness specific biomolecular interactions suffer from issues related to protein denaturation, contamination and the need to maintain biomolecule-specific conditions for efficient protein capture. Here, we report a versatile and scalable method for the purification of recombinant protein therapeutics. The method exploits the high-affinity and controllable host-guest interactions between cucurbit[7]uril (CB[7]) and selected guests such as adamantylammonium. We show that the Herceptin (the brand name of trastuzumab, a monoclonal antibody drug used to treat breast cancer) and the much smaller cytokine interferon α-2a can be purified by site-specifically tagging them with adamantylammonium using the enzyme sortase A, followed by high-affinity binding with CB[7]-conjugated agarose beads and the recovery of the protein using a guest with a stronger affinity for CB[7]. The thermal and chemical stability of CB[7] beads and their scalability, recyclability and low cost may also make them advantageous for the manufacturing of biosimilars.

Bio-orthogonal Supramolecular Latching inside Live Animals and Its Application for in Vivo Cancer Imaging.

Here, we demonstrate a supramolecular latching tool for bio-orthogonal noncovalent anchoring of small synthetic molecules in live animal models using a fully synthetic high-affinity binding pair between cucurbit[7]uril (CB[7]) and adamantylammonium (AdA). This supramolecular latching system is small (∼1 kDa), ensuring efficient uptake into cells, tissues, and whole organisms. It is also chemically robust and resistant to enzymatic degradation and analogous to well-characterized biological systems in terms of noncovalent binding. Occurrence of fluorescence resonance energy transfer (FRET) between cyanine 3-CB[7] (Cy3-CB[7]) and boron-dipyrromethene 630/650X-AdA (BDP630/650-AdA) inside a live worm (Caenorhabditis elegans) indicates efficient in situ high-affinity association between AdA and CB[7] inside live animals. In addition, selective visualization of a cancer site of a live mouse upon supramolecular latching of cyanine 5-AdA (Cy5-AdA) on prelocalized CB[7]-conjugating antibody on the cancer site demonstrates the potential of this synthetic system for in vivo cancer imaging. These findings provide a fresh insight into the development of new chemical biology tools and medical therapeutic systems.

Cucurbit[n]uril-based amphiphiles that self-assemble into functional nanomaterials for therapeutics.

Some host-guest complexes of cucurbit[n]uril (CB[n]) host molecules act as supramolecular amphiphiles (SAs), which hierarchically self-assemble into various nanomaterials such as vesicles, micelles, nanorods, and nanosheets in water. The structures and functions of the nanomaterials can be controlled by supramolecular engineering of the host-guest complexes. In addition, functionalization at the periphery of CB[6] and CB[7] generates CB[n]-based molecular amphiphiles (MAs) that can also self-assemble into vesicles or micelle-like nanoparticles in water. Taking advantage of the molecular cavities of CBs and their strong guest recognition properties, the surface of the self-assembled nanomaterials can be easily decorated with various functional tags in a non-covalent manner. In this feature article, the two types (SAs and MAs) of CB-based amphiphiles, their self-assemblies and their applications for nanotherapeutics and theranostics are presented with future perspectives.

Tumor vasodilation by N-Heterocyclic carbene-based nitric oxide delivery triggered by high-intensity focused ultrasound and enhanced drug homing to tumor sites for anti-cancer therapy.

Nitric oxide (NO) is widely known as an effective vasodilator at low concentrations. Drug delivery systems combined with NO can dilate blood vessels surrounding tumor tissues, and the drug accumulation in tumors is accelerated by the enhanced permeability and retention effect, leading to an improvement in the anti-tumor effect. N-heterocyclic carbene-based NO donors (e.g., 1,3-bis-(2,4,6-trimethylphenyl)imidazolylidene nitric oxide (IMesNO) have been developed for stable NO storing in air and water, and NO release by thermolysis. Herein, we demonstrated on-demand NO release by high-intensity focused ultrasound (HIFU) as a stimulus, which generated high heat and exerted an ablation effect when treated in vivo. We demonstrated IMesNO to be a HIFU-responsive NO donor and its potential application in vivo using IMesNO-loaded micelles. Moreover, IMesNO-loaded micelles mixed with drug-loaded micelles (IMesNO/DOX@MCs) showed acceleration of drug accumulation in tumor sites and enhanced tumor growth inhibition. Thus, our findings suggest a potential clinical bioapplication of NO-releasing drug-loaded micelles owing to the therapeutic function of NO and HIFU treatment for anti-cancer therapy.

Cucurbit[7]uril-conjugated dyes as live cell imaging probes: investigation on their cellular uptake and excretion pathways.

Here we report the endocytosis and excretion pathways of two different dye-conjugated cucurbit[7]urils, (cyanine 3-conjugated CB[7] and rhodamine X-conjugated CB[7]), which have great potential as molecular probes for live cell imaging. The dye-CB[7]s are translocated into live cells (human breast carcinoma cells, MCF-7) via multiple pathways, predominantly by clathrin-mediated endocytosis, and excreted from cells via lysosome-associated exocytosis. Interestingly, the CB[7] moiety has a substantial influence on the uptake and excretion pathways. These findings may widen the applications of the dyes conjugated to CB[7] and assist in the design of new molecular probes for live cell imaging.

Iron Porphyrins Embedded into a Supramolecular Porous Organic Cage for Electrochemical CO2 Reduction in Water.

A porous organic cage composed of six iron tetraphenylporphyrins was used as a supramolecular catalyst for electrochemical CO2 -to-CO conversion. This strategy enhances active site exposure and substrate diffusion relative to the monomeric catalyst, resulting in CO generation with near-quantitative Faradaic efficiency in pH 7.3 water, with activities reaching 55 250 turnovers. These results provide a starting point for the design of supramolecular catalysts that can exploit the properties of the surrounding matrix yet retain the tunability of the original molecular unit.

Separation of Acetylene from Carbon Dioxide and Ethylene by a Water-Stable Microporous Metal-Organic Framework with Aligned Imidazolium Groups inside the Channels.

Separation of acetylene from carbon dioxide and ethylene is challenging in view of their similar sizes and physical properties. Metal-organic frameworks (MOFs) in general are strong candidates for these separations owing to the presence of functional pore surfaces that can selectively capture a specific target molecule. Here, we report a novel 3D microporous cationic framework named JCM-1. This structure possesses imidazolium functional groups on the pore surfaces and pyrazolate as a metal binding group, which is well known to form strong metal-to-ligand bonds. The selective sorption of acetylene over carbon dioxide and ethylene in JCM-1 was successfully demonstrated by equilibrium gas adsorption analysis as well as dynamic breakthrough measurement. Furthermore, its excellent hydrolytic stability makes the separation processes highly recyclable without a substantial loss in acetylene uptake capacity.

Mono-allyloxylated Cucurbit[7]uril Acts as an Unconventional Amphiphile To Form Light-Responsive Vesicles.

Serendipitously, mono-allyloxylated cucurbit[7]uril (AO1 CB[7]) was discovered to act as an unconventional amphiphile which self-assembles into light-responsive vesicles (AO1 CB[7]VC) in water. Although the mono-allyloxy group, directly tethered on the periphery of CB[7], is much shorter (C4) than the hydrophobic tails of conventional amphiphiles, it played an important role in vesicle formation. Light-activated transformation of the allyloxy group by conjugation with glutathione was exploited as a remote tool to disrupt the vesicle. The vesicle showed on-demand release of cargo upon irradiation by a laser, after they were internalized into cancer cells. This result demonstrated the potential of AO1 CB[7]VC as a new type of light-responsive intracellular delivery vehicle for the release of therapeutic cargo, within cells, on demand.

Oxime Ether Radical Cations Stabilized by N-Heterocyclic Carbenes.

N-heterocyclic carbene (NHC) nitric oxide (NHCNO) radicals, which can be regarded as iminoxyl radicals stabilized by NHCs, were found to react with a series of silyl and alkyl triflates to generate the corresponding oxime ether radical cations. The structures of the resulting oxime ether radical cations were determined by X-ray crystallography, along with EPR and computational analysis. In contrast, lutidinium triflate produced a 1:1 mixture of [NHCNO+ ][OTf- ] and [NHCNHOH+ ][OTf- ] upon the reaction with NHCNO. This study adds an important example of stable singlet carbenes for stabilizing main-group radicals because of their π-conjugating effect, the synthesis and structures of which have not been reported previously.

Autophagy Caught in the Act: A Supramolecular FRET Pair Based on an Ultrastable Synthetic Host-Guest Complex Visualizes Autophagosome-Lysosome Fusion.

A supramolecular FRET pair based on the ultrahigh binding affinity between cyanine 3 conjugated cucurbit[7]uril (CB[7]-Cy3) and cyanine 5 conjugated adamantylamine (AdA-Cy5) was exploited as a new synthetic tool for imaging cellular processes in live cells. Confocal laser scanning microscopy revealed that CB[7]-Cy3 and AdA-Cy5 were intracellularly translocated and accumulated in lysosomes and mitochondria, respectively. CB[7]-Cy3 and AdA-Cy5 then formed a host-guest complex, reported by a FRET signal, as a result of the fusion of lysosomes and mitochondria. This observation not only indicated that CB[7] forms a stable complex with AdA in a live cell, but also suggested that this FRET pair can visualize dynamic organelle fusion processes, such as those involved in the degradation of mitochondria through autophagy (mitophagy), by virtue of its small size, chemical stability, and ease of use.

Nanoscale Control of Amyloid Self-Assembly Using Protein Phase Transfer by Host-Guest Chemistry.

Amyloid fibrils have recently been highlighted for their diverse applications as functional nanomaterials in modern chemistry. However, tight control to obtain a targeted fibril length with low heterogeneity has not been achieved because of the complicated nature of amyloid fibrillation. Herein, we demonstrate that fibril assemblies can be homogeneously manipulated with desired lengths from ~40 nm to ~10 µm by a phase transfer of amyloid proteins based on host-guest chemistry. We suggest that host-guest interactions with cucurbit[6]uril induce a phase transfer of amyloid proteins (human insulin, human islet amyloid polypeptide, hen egg lysozyme, and amyloid-ß 1-40 & 1-42) from the soluble state to insoluble state when the amount of cucurbit[6]uril exceeds its solubility limit in solution. The phase transfer of the proteins kinetically delays the nucleation of amyloid proteins, while the nuclei formed in the early stage are homogeneously assembled to fibrils. Consequently, supramolecular assemblies of amyloid proteins with heterogeneous kinetics can be controlled by protein phase transfer based on host-guest interactions.

Enrichment of Specifically Labeled Proteins by an Immobilized Host Molecule.

Chemical proteomics relies primarily on click-chemistry-based protein labeling and biotin-streptavidin enrichment, but these techniques have inherent limitations. Enrichment of intracellular proteins using a totally synthetic host-guest complex is described, overcoming the problem associated with the classical approach. We achieve this by affinity-based protein labeling with a target-specific probe molecule conjugated to a high-affinity guest (suberanilohydroxamic acid-ammonium-adamantane; SAHA-Ad) and then enriching the labeled species using a cucurbit[7]uril bead. This method shows high specificity for labeled molecules in a MDA-MB-231 breast cancer cell lysate. Moreover, this method shows promise for labeling proteins in live cells.

Three-dimensional bioprinting of multilayered constructs containing human mesenchymal stromal cells for osteochondral tissue regeneration in the rabbit knee joint.

The use of cell-rich hydrogels for three-dimensional (3D) cell culture has shown great potential for a variety of biomedical applications. However, the fabrication of appropriate constructs has been challenging. In this study, we describe a 3D printing process for the preparation of a multilayered 3D construct containing human mesenchymal stromal cells with a hydrogel comprised of atelocollagen and supramolecular hyaluronic acid (HA). This construct showed outstanding regenerative ability for the reconstruction of an osteochondral tissue in the knee joints of rabbits. We found that the use of a mechanically stable, host-guest chemistry-based hydrogel was essential and allowed two different types of extracellular matrix (ECM) hydrogels to be easily printed and stacked into one multilayered construct without requiring the use of potentially harmful chemical reagents or physical stimuli for post-crosslinking. To the best of our knowledge, this is the first study to validate the potential of a 3D printed multilayered construct consisting of two different ECM materials (atelocollagen and HA) for heterogeneous tissue regeneration using an in vivo animal model. We believe that this 3D printing-based platform technology can be effectively exploited for regeneration of various heterogeneous tissues as well as osteochondral tissue.

Genetically engineered mesenchymal stem cell therapy using self-assembling supramolecular hydrogels.

Stem cell therapy has attracted a great deal of attention for treating intractable diseases such as cancer, stroke, liver cirrhosis, and ischemia. Especially, mesenchymal stem cells (MSCs) have been widely investigated for therapeutic applications due to the advantageous characteristics of long life-span, facile isolation, rapid proliferation, prolonged transgene expression, hypo-immunogenicity, and tumor tropism. MSCs can exert their therapeutic effects by releasing stress-induced therapeutic molecules after their rapid migration to damaged tissues. Recently, to improve the therapeutic efficacy, genetically engineered MSCs have been developed for therapeutic transgene expression by viral gene transduction and non-viral gene transfection. In general, the number of therapeutic cells for injection should be more than several millions for effective cell therapy. Adequate carriers for the controlled delivery of MSCs can reduce the required cell numbers and extend the duration of therapeutic effect, which provide great benefits for chronic disease patients. In this review, we describe genetic engineering of MSCs, recent progress of self-assembling supramolecular hydrogels, and their applications to cell therapy for intractable diseases and tissue regeneration.

Self-assembly of nanostructured materials through irreversible covalent bond formation.

Over the past decades, numerous efforts have been devoted to synthesizing nanostructured materials with specific morphology because their size and shape play an important role in determining their functions. Self-assembly using weak and reversible interactions or bonds has provided synthetic routes toward various nanostructures because it allows a "self-checking" and "self-error-correcting" process under thermodynamic control. By contrast, the use of irreversible covalent bonds, despite the potential to generate more robust structures, has been disfavored in the synthesis of well-defined nanomaterials largely due to the lack of such self-error-correcting mechanisms. To date, the use of irreversible bonds is largely limited to covalent fixation of preorganized building blocks on a template, which, though capable of producing shape-persistent and robust nanostructured materials, often requires a laborious and time-consuming multistep processes. Constructing well-defined nanostructures by self-assembly using irreversible covalent bonds without help of templates or preorganization of components remains a challenge. This Account describes our recent discoveries and progress in self-assembly of nanostructured materials through strong, practically irreversible covalent bond formation and their applications in various areas including drug delivery, anticancer therapy, and heterogeneous catalysis. The key to the success of this approach is the use of rationally designed building blocks possessing multiple in-plane reactive groups at the periphery. These blocks can then successfully grow into flat oligomeric patches through irreversible covalent bond formation without the aid of preorganization or templates. Further growth of the patches with or without curvature generation drives the system to the formation of polymer nanocapsules, two-dimensional (2D) polymer films, and toroidal nanotubular microrings. Remarkably, the final morphology can be specified by a few simple parameters: the reaction medium, bending rigidity of the system, and orientation of the reactive groups. Theoretical studies support the spontaneous formation of such nanostructured materials in terms of energetics and successfully predict or explain their size distributions. Although the lack of self-error-correcting mechanisms results in defect sites in these nanostructures, the high efficiency and relative simplicity of our novel approach demonstrates the potential power of using irreversible covalent bonds to generate a diverse range of shape-persistent and robust nanostructures that is likely to enrich the repertoire of self-assembled nanomaterials.

Supramolecular hydrogels for long-term bioengineered stem cell therapy.

Synthetic hydrogels have been extensively investigated as artificial extracellular matrices (ECMs) for tissue engineering in vitro and in vivo. Crucial challenges for such hydrogels are sustaining long-term cytocompatible encapsulation and providing appropriate cues at the right place and time for spatio-temporal control of the cells. Here, in situ supramolecularly assembled and modularly modified hydrogels for long-term engineered mesenchymal stem cell (eMSC) therapy are reported using cucurbit[6]uril-conjugated hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug-conjugated CB[6] (drug-CB[6]). The eMSCs producing enhanced green fluorescence protein (EGFP) remain alive and emit the fluorescence within CB[6]/DAH-HA hydrogels in mice for more than 60 d. Furthermore, the long-term expression of mutant interleukin-12 (IL-12M) by eMSCs within the supramolecular hydrogels results in effective inhibition of tumor growth with a significantly enhanced survival rate. Taken together, these findings confirm the feasibility of supramolecular HA hydrogels as 3D artificial ECMs for cell therapies and tissue engineering applications.

Highly stable, water-dispersible metal-nanoparticle-decorated polymer nanocapsules and their catalytic applications.

A facile synthesis of highly stable, water-dispersible metal-nanoparticle-decorated polymer nanocapsules (M@CB-PNs: M=Pd, Au, and Pt) was achieved by a simple two-step process employing a polymer nanocapsule (CB-PN) made of cucurbit[6]uril (CB[6]) and metal salts. The CB-PN serves as a versatile platform where various metal nanoparticles with a controlled size can be introduced on the surface and stabilized to prepare new water-dispersible nanostructures useful for many applications. The Pd nanoparticles on CB-PN exhibit high stability and dispersibility in water as well as excellent catalytic activity and recyclability in carbon-carbon and carbon-nitrogen bond-forming reactions in aqueous medium suggesting potential applications as a green catalyst.

Supramolecular inhibition of amyloid fibrillation by cucurbit[7]uril.

Amyloid fibrils are insoluble protein aggregates comprised of highly ordered ß-sheet structures and they are involved in the pathology of amyloidoses, such as Alzheimer's disease. A supramolecular strategy is presented for inhibiting amyloid fibrillation by using cucurbit[7]uril (CB[7]). CB[7] prevents the fibrillation of insulin and ß-amyloid by capturing phenylalanine (Phe) residues, which are crucial to the hydrophobic interactions formed during amyloid fibrillation. These results suggest that the Phe-specific binding of CB[7] can modulate the intermolecular interaction of amyloid proteins and prevent the transition from monomeric to multimeric states. CB[7] thus has potential for the development of a therapeutic strategy for amyloidosis.