Cell-Compatible Nanoprobes for Imaging Intracellular Phosphatase Activities.

Phosphatases play an important role in cell biology, but only a few probes are suitable for selectively imaging phosphatase activity in live cells, because the current probes require cell fixation or exhibit considerable cytotoxicity. Herein, we show that conjugating a d-peptide to a quinazolinone derivative generates cell-compatible, biostable probes for imaging the phosphatase activity inside live cells. Moreover, our results show that inhibiting ectophosphatases is a critical factor for imaging intracellular phosphatases. As the first example of using selective inhibitors to ensure intracellular function of molecular probes, this work illustrates a facile approach to design molecular probes for profiling the activities of enzymes in a spatial, selective manner in a complicated environment.

Cellular Uptake of A Taurine-Modified, Ester Bond-Decorated D-Peptide Derivative via Dynamin-Based Endocytosis and Macropinocytosis.

Most of the peptides used for promoting cellular uptake bear positive charges. In our previous study, we reported an example of taurine (bearing negative charges in physiological conditions) promoting cellular uptake of D-peptides. Taurine, conjugated to a small D-peptide via an ester bond, promotes the cellular uptake of this D-peptide. Particularly, intracellular carboxylesterase (CES) instructs the D-peptide to self-assemble and to form nanofibers, which largely disfavors efflux and further enhances the intracellular accumulation of the D-peptide, as supported by that the addition of CES inhibitors partially impaired cellular uptake of this molecule in mammalian cell lines. Using dynamin 1, 2, and 3 triple knockout (TKO) mouse fibroblasts, we demonstrated that cells took up this molecule via macropinocytosis and dynamin-dependent endocytosis. Imaging of Drosophila larval blood cells derived from endocytic mutants confirmed the involvement of multiple endocytosis pathways. Electron microscopy (EM) indicated that the precursors can form aggregates on the cell surface to facilitate the cellular uptake via macropinocytosis. EM also revealed significantly increased numbers of vesicles in the cytosol. This work provides new insights into the cellular uptake of taurine derivative for intracellular delivery and self-assembly of D-peptides.

Adaptive Multifunctional Supramolecular Assemblies of Glycopeptides Rapidly Enable Morphogenesis.

Despite the well-established biophysical principle of adhesion-guided in vitro morphogenesis, there are few single synthetic molecular species that can rapidly enable morphogenesis (e.g., a cell monolayer to cell spheroids) in a cell culture because adhesion inherently involves many signals. Here we show the use of adaptive multifunctional supramolecular assemblies of glycopeptides, consisting of cell adhesion sequence and saccharide, to induce cell spheroids rapidly from a monolayer of cells. Having a general architecture of N-terminal capping, glycosylation, and an integrin-binding sequence, the glycopeptides self-assemble to form a dynamic continuum of nanostructures (i.e., from nanoparticles to nanofibers) to affect the interactions of integrins, E-selectin, and cadherins with their natural ligands and to act adaptively according to the cellular environment. Such adaptive (i.e., context-dependent) interactions weaken cell-substratum adhesion and enhance intercellular interactions, which rapidly and transiently induce cell spheroids. This work illustrates the use of supramolecular assemblies of simple glycopeptides to modulate biophysical conditions for regulating cell functions, which is a new approach for developing biomaterials.

Enzymatic Self-Assembly Confers Exceptionally Strong Synergism with NF-κB Targeting for Selective Necroptosis of Cancer Cells.

As a promising molecular process for selectively inhibiting cancer cells without inducing acquired drug resistance, enzyme-instructed self-assembly (EISA) usually requires relatively high dosages. Despite its discovery 30 years ago, the translation of the knowledge about NF-κB signaling into clinic remains complicated due to the broad roles of NF-κB in cellular regulation. Here we show that integrating EISA and NF-κB targeting boosts the efficacy of EISA over an order of magnitude without compromising selectivity against cancer cells. That is, in situ enzymatic self-assembly of a tetrapeptide results in nanofibers, which hardly affect cell viability, but lead to inductive expression of tumor necrosis factor receptor 2 (TNFR2) and decreased expression of three key proteins at the upstream of NF-κB pathway in the cancer cells. Adding the inhibitors targeting NF-κB further decreases the expressions of those upstream proteins, which turns the otherwise innocuous nanofibers to being lethal to the cancer cells, likely causing necroptosis. As the first case of using supramolecular processes to enable synthetic lethality, this work illustrates a versatile approach to translate key regulatory circuits into promising therapeutic targets.

Selection of Secondary Structures of Heterotypic Supramolecular Peptide Assemblies by an Enzymatic Reaction.

In a model study to investigate the consequence of reactions of intrinsically disordered regions (IDRs) of proteins in the context of the formation of highly ordered structures, we found that enzymatic reactions control the secondary structures of peptides during assembly. Specifically, phosphorylation of an α-helix-dominant peptide results in mostly disordered conformations, which become ß-strand-dominant after enzymatic dephosphorylation to regenerate the peptide. In the presence of another peptide largely with a ß-strand conformation, direct coassembly of the peptides results in amorphous aggregates consisting of α-helix and ß-strand peptides, but the enzymatically generated peptide coassemblies (from the phosphopeptide) mainly adopt a ß-strand conformation and form ordered structures (e.g., nanofibers). These results indicate that enzymatic dephosphorylation instructs conformationally flexible peptides to adopt thermodynamically favorable conformations in homotypic or heterotypic supramolecular assemblies.

Aromatic-Aromatic Interactions Enable α-Helix to ß-Sheet Transition of Peptides to Form Supramolecular Hydrogels.

Isolated short peptides usually are unable to maintain their original secondary structures due to the lack of the restriction from proteins. Here we show that two complementary pentapeptides from a ß-sheet motif of a protein, being connected to an aromatic motif (i.e., pyrene) at their C-terminal, self-assemble to form ß-sheet like structures upon mixing. Besides enabling the self-assembly to result in supramolecular hydrogels upon mixing, aromatic-aromatic interactions promote the pentapeptides transform from α-helix to ß-sheet conformation. As the first example of using aromatic-aromatic interactions to mimic the conformational restriction in a protein, this work illustrates a bioinspired way to generate peptide nanofibers with predefined secondary structures of the peptides by a rational design using protein structures as the blueprint.

Enzymatic self-assembly of an immunoreceptor tyrosine-based inhibitory motif (ITIM).

Here we show the first example of an immunoreceptor tyrosine-based inhibitory motif (ITIM), LYYYYL, as well as its enantiomeric or retro-inverso peptide, to self-assemble in water via enzyme-instructed self-assembly. Upon enzymatic dephosphorylation, the phosphohexapeptides become hexapeptides, which self-assemble in water to result in supramolecular hydrogels. This work illustrates a new approach to design bioinspired soft materials from a less explored, but important pool of immunomodulatory peptides.

Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials.

In this review we intend to provide a relatively comprehensive summary of the work of supramolecular hydrogelators after 2004 and to put emphasis particularly on the applications of supramolecular hydrogels/hydrogelators as molecular biomaterials. After a brief introduction of methods for generating supramolecular hydrogels, we discuss supramolecular hydrogelators on the basis of their categories, such as small organic molecules, coordination complexes, peptides, nucleobases, and saccharides. Following molecular design, we focus on various potential applications of supramolecular hydrogels as molecular biomaterials, classified by their applications in cell cultures, tissue engineering, cell behavior, imaging, and unique applications of hydrogelators. Particularly, we discuss the applications of supramolecular hydrogelators after they form supramolecular assemblies but prior to reaching the critical gelation concentration because this subject is less explored but may hold equally great promise for helping address fundamental questions about the mechanisms or the consequences of the self-assembly of molecules, including low molecular weight ones. Finally, we provide a perspective on supramolecular hydrogelators. We hope that this review will serve as an updated introduction and reference for researchers who are interested in exploring supramolecular hydrogelators as molecular biomaterials for addressing the societal needs at various frontiers.

Hyper-Crosslinkers Lead to Temperature- and pH-Responsive Polymeric Nanogels with Unusual Volume Change.

Hydrogels consisting of carboxylic acid groups and N-isopropylacrylamide as pendants on their polymeric network usually exhibit volume expansion upon deprotonation or volume contraction when being heated. Demonstrated here is an anti-intuitive case of a hydrogel containing multiple carboxylic acid groups at each crosslinking point in the polymeric network, which shrinks upon increasing pH from 1 to 7 at 37 °C or expands upon heating from 25 to 37 °C at pH 1. The unexpected volume change originates from the high percentage of the crosslinker in the polymers, as detected by solid-state 13 C NMR spectroscopy. In addition, the volume changes are thermally reversible. As the first example of the use of functional hyper-crosslinkers to control the pH and thermal responses of nanogels, this work illustrates a new way to design soft materials with unusual behaviors.

Enzyme-Instructed Self-Assembly of Small D-Peptides as a Multiple-Step Process for Selectively Killing Cancer Cells.

Selective inhibition of cancer cells remains a challenge in chemotherapy. Here we report the molecular and cellular validation of enzyme-instructed self-assembly (EISA) as a multiple step process for selectively killing cancer cells that overexpress alkaline phosphatases (ALPs). We design and synthesize two kinds of D-tetrapeptide containing one or two phosphotyrosine residues and with the N-terminal capped by a naphthyl group. Upon enzymatic dephosphorylation, these D-tetrapeptides turn into self-assembling molecules to form nanofibers in water. Incubating these D-tetrapeptides with several cancer cell lines and one normal cell line, the unphosphorylated D-tetrapeptides are innocuous to all the cell lines, the mono- and diphosphorylated D-tetrapeptides selectively inhibit the cancer cells, but not the normal cell. The monophosphorylated D-tetrapeptides exhibit more potent inhibitory activity than the diphosphorylated D-tetrapeptides do; the cancer cell lines express higher level of ALPs are more susceptible to inhibition by the phosphorylated D-tetrapeptides; the precursors of D-tetrapeptides that possess higher self-assembling abilities exhibit higher inhibitory activities. These results confirm the important role of enzymatic reaction and self-assembly. Using uncompetitive inhibitors of ALPs and fluorescent D-tetrapeptides, we delineate that the enzyme catalyzed dephosphorylation and the self-assembly steps, together, result in the localization of the nanofibers of D-tetrapeptides for killing the cancer cells. We find that the cell death modality likely associates with the cell type and prove the interactions between nanofibers and the death receptors. This work illustrates a paradigm-shifting and biomimetic approach and contributes useful molecular insights for the development of spatiotemporal defined supramolecular processes/assemblies as potential anticancer therapeutics.

Ligand-Receptor Interaction Modulates the Energy Landscape of Enzyme-Instructed Self-Assembly of Small Molecules.

The concurrence of enzymatic reaction and ligand-receptor interactions is common for proteins, but rare for small molecules and has yet to be explored. Here we show that ligand-receptor interaction modulates the morphology of molecular assemblies formed by enzyme-instructed assembly of small molecules. While the absence of ligand-receptor interaction allows enzymatic dephosphorylation of a precursor to generate the hydrogelator that self-assembles to form long nanofibers, the presence of the ligand-receptor interaction biases the pathway to form precipitous aggregates containing short nanofibers. While the hydrogelators self-assemble to form nanofibers or nanoribbons that are unable to bind with the ligand (i.e., vancomycin), the addition of surfactant breaks up the assemblies to restore the ligand-receptor interaction. In addition, an excess amount of the ligands can disrupt the nanofibers and result in the precipitates. As the first example of the use of ligand-receptor interaction to modulate the kinetics of enzymatic self-assembly, this work not only provides a solution to evaluate the interaction between aggregates and target molecules but also offers new insight for understanding the emergent behavior of sophisticated molecular systems having multiple and parallel processes.

Regulating the Rate of Molecular Self-Assembly for Targeting Cancer Cells.

Besides tight and specific ligand-receptor interactions, the rate regulation of the formation of molecular assemblies is one of fundamental features of cells. But the latter receives little exploration for developing anticancer therapeutics. Here we show that a simple molecular design of the substrates of phosphatases-tailoring the number of phosphates on peptidic substrates-is able to regulate the rate of molecular self-assembly of the enzyme reaction product. Such a rate regulation allows selective inhibition of osteosarcoma cells over hepatocytes, which promises to target cancer cells in a specific organ. Moreover, our result reveals that the direct measurement of the rate of the self-assembly in a cell-based assay provides precise assessment of the cell targeting capability of self-assembly. This work, as the first report establishing rate regulation of a multiple-step process to inhibit cells selectively, illustrates a fundamentally new approach for controlling the fate of cells.

Supramolecular Glycosylation Accelerates Proteolytic Degradation of Peptide Nanofibrils.

Despite the recent consensus that the oligomers of amyloid peptides or aberrant proteins are cytotoxic species, there is still a need for an effective way to eliminate the oligomers. Based on the fact that normal proteins are more glycosylated than pathogenic proteins, we show that a conjugate of nucleobase, peptide, and saccharide binds to peptides from molecular nanofibrils and accelerates the proteolytic degradation of the molecular nanofibrils. As the first example of the use of supramolecular glycosylation to dissociate molecular nanofibrils and to accelerate the degradation of peptide aggregates, this work illustrates a new method that ultimately may lead to an effective approach for degrading cytotoxic oligomers of peptides or aberrant proteins.

Taurine Boosts Cellular Uptake of Small D-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly.

Due to their biostability, D-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, D-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small D-peptides in mammalian cells by >10-fold, from 118 µM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and D-peptide allows intracellular esterase to trigger intracellular self-assembly of the D-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly.

Ligand-receptor interaction catalyzes the aggregation of small molecules to induce cell necroptosis.

Because they exhibit important biological functions, from unfolding proteins to activating enzymes to controlling cell fates, aggregates of small molecules are able to serve as functional molecular entities in cellular environments. However, the inability to precisely control their production has hampered the understanding and exploration of their biological functions. Here we show that the well-established ligand-receptor interaction between vancomycin and d-Ala-d-Ala catalyzes the aggregation of a d-Ala-d-Ala-containing small peptide derivative in water. The resulting aggregates largely adhere to the cell surface to induce cell necroptosis. Mutation of d-Ala-d-Ala to l-Ala-l-Ala or removal of the aromatic group in the derivative results in innocuous compounds, confirming that the aromatic-aromatic and ligand-receptor interactions are responsible for the formation and corresponding cytotoxicity of the aggregates. In addition to being the first example of ligand-receptor interaction-catalyzed aggregation of small molecules on the surface of mammalian cells, this work provides useful insights for understanding the cytotoxicity of molecular aggregates of small molecules.

Supramolecular Detoxification of Neurotoxic Nanofibrils of Small Molecules via Morphological Switch.

Insoluble amyloid plagues are likely cytoprotective, but the cellular mechanism remains less known. To model ß-amyloid we use a small peptide derivative to generate cytotoxic nanofibrils that cause the death of model neuron cells (i.e., PC12). The use of supramolecular interaction effectively converts the nanofibrils to nanoparticles that are innocuous to cells. This approach also removes the cytotoxicity of the fibrils to other mammalian cells (e.g., HeLa). Preliminary mechanistic study reveals that, in contrast to the fibrils, the particles promote the expression of TNFR2, a cell survival signal, and decrease the expression of TNFR1 and DR5, two extrinsic cell death receptors. As the first use of ligand-receptor interaction to abrogate the cytotoxicity of nanoscale assemblies of small molecules, this work illustrates an effective way to use supramolecular interaction to control the morphology of supramolecular assemblies for modulating their biological activity.

Mixing biomimetic heterodimers of nucleopeptides to generate biocompatible and biostable supramolecular hydrogels.

As a new class of biomaterials, most supramolecular hydrogels formed by small peptides require the attachment of long alkyl chains, multiple aromatic groups, or strong electrostatic interactions. Based on the fact that the most abundant protein assemblies in nature are dimeric, we select short peptide sequences from the interface of a heterodimer of proteins with known crystal structure to conjugate with nucleobases to form nucleopeptides. Being driven mainly by hydrogen bonds, the nucleopeptides self-assemble to form nanofibers, which results in supramolecular hydrogels upon simple mixing of two distinct nucleopeptides in water. Moreover, besides being biocompatible to mammalian cells, the heterodimer of the nucleopeptides exhibit excellent proteolytic resistance against proteinase K. This work illustrates a new and rational approach to create soft biomaterials by a supramolecular hydrogelation triggered by mixing heterodimeric nucleopeptides.

Synthesis and evaluation of the biostability and cell compatibility of novel conjugates of nucleobase, peptidic epitope, and saccharide.

This article reports the synthesis of a new class of conjugates containing a nucleobase, a peptidic epitope, and a saccharide and the evalution of their gelation, biostability, and cell compatibility. We demonstrate a facile synthetic process, based on solid-phase peptide synthesis of nucleopeptides, to connect a saccharide with the nucleopeptides for producing the target conjugates. All the conjugates themselves (1-8) display excellent solubility in water without forming hydrogels. However, a mixture of 5 and 8 self-assembles to form nanofibers and results in a supramolecular hydrogel. The proteolytic stabilities of the conjugates depend on the functional peptidic epitopes. We found that TTPV is proteolytic resistant and LGFNI is susceptible to proteolysis. In addition, all the conjugates are compatible to the mammalian cells tested.

Aromatic-aromatic interactions enhance interfiber contacts for enzymatic formation of a spontaneously aligned supramolecular hydrogel.

Anisotropy or alignment is a critical feature of functional soft materials in living organisms, but it remains a challenge for spontaneously generating anisotropic gel materials. Here we report a molecular design that increases intermolecular aromatic-aromatic interactions of hydrogelators during enzymatic hydrogelation for spontaneously forming an anisotropic hydrogel. This process, relying on both aromatic-aromatic interactions and enzyme catalysis, results in spontaneously aligned supramolecular nanofibers as the matrices of a monodomain hydrogel that exhibits significant birefringence. This work, as the first example of monodomain hydrogels formed via an enzymatic reaction, illustrates a new biomimetic approach for generating aligned anisotropic soft materials.

Enzymatic transformation of phosphate decorated magnetic nanoparticles for selectively sorting and inhibiting cancer cells.

As an important and necessary step of sampling biological specimens, the separation of malignant cells from a mixed population of cells usually requires sophisticated instruments and/or expensive reagents. For health care in the developing regions, there is a need for an inexpensive sampling method to capture tumor cells for rapid and accurate diagnosis. Here we show that an underexplored generic difference-overexpression of ectophosphatases-between cancer and normal cells triggers the d-tyrosine phosphate decorated magnetic nanoparticles (Fe3O4-p(d-Tyr)) to adhere selectively on cancer cells upon catalytic dephosphorylation, which enables magnetic separation of cancer cells from mixed population of cells (e.g., cocultured cancer cell (HeLa-GFP) and stromal cells (HS-5)). Moreover, the Fe3O4-p(d-Tyr) nanoparticles also selectively inhibit cancer cells in the coculture. As a general method to broadly target cancer cells without highly specific ligand-receptor interactions (e.g., antibodies), the use of an enzymatic reaction to spatiotemporally modulate the state of various nanostructures in cellular environments will ultimately lead to the development of new theranostic applications of nanomaterials.