Chiral Supramolecular Nanofibers Regulated Tumor-Derived Exosomes Secretion for Constructing an Anti-Tumor Extracellular Microenvironment.

Tumor-derived exosomes (TDEs) induced extracellular microenvironment has recently been validated to be critical for tumor progression and metastasis, however, remodeling it for oncotherapy still remains a major challenge due to difficulty in regulation of TDEs secretion. Herein, the supramolecular chiral nanofibers, composed of L/D-phenylalanine derivates (L/D-Phe) and linear hyaluronic acid (HA), are successfully employed to construct TDEs induced anti-tumor extracellular microenvironment. The left-handed L-Phe @HA nanofibers significantly inhibit TDEs secretion into extracellular microenvironment, which results in suppression of tumor proliferation and metastasis in vitro and vivo. Biological assays and theoretical modeling reveal that these results are mainly attributed to strong adsorption of the key exosomes transporters (Ras-related protein Rab-27A and synaptosome-associated protein 23) on left-handed L-Phe @HA nanofibers via enhanced stereoselective interaction, leading to degradation and phosphorylated dropping of exosomes transporters. Subsequently, transfer function of exosomes transporters is limited, which causes remarkable inhibition of TDEs secretion. These findings provide a promising novel insight of chiral functional materials to establish an anti-tumor extracellular microenvironment via regulation of TDEs secretion.

Assembly of Helical Nanostructures: Solvent-Induced Morphology Transition and Its Effect on Cell Adhesion.

Being able to precisely manipulate both the morphology and chiroptical signals of supramolecular assemblies will help to better understand the natural biological self-assembly mechanism. Two simple l/d-phenylalanine-based derivatives (L/DPFM) have been designed, and their solvent-dependent morphology evolutions are illustrated. It was found that, as the content of H2 O in aqueous ethanol solutions was increased, LPFM self-assembles first into right-handed nanofibers, then flat fibrous structures, and finally inversed left-handed nanofibers. Assemblies in ethanol and H2 O exhibit opposite conformations and circular dichroism (CD) signals even though they are constructed from the same molecules. Thus, the morphology-dependent cell adhesion and proliferation behaviors are further characterized. Left-handed nanofibers are found to be more favorable for cell adhesion than right-handed nanostructures. Quantitative AFM analysis showed that the L929 cell adhesion force on left-handed LPFM fibers is much higher than that on structures with inversed handedness. Moreover, the value of cell Young's modulus is lower for left-handed nanofibrous films, which indicates better flexibility. The difference in cell-substrate interactions might lead to different effects on cell behavior.

Realizing Abundant Chirality Inversion of Supramolecular Nanohelices by Multiply Manipulating the Binding Sites in Molecular Blocks.

The induction of diverse chirality regulation in nature by multiple binding sites of biomolecules is ubiquitous and plays an essential role in determining the biofunction of biosystems. However, mimicking this biological phenomenon and understanding at a molecular level its mechanism with the multiple binding sites by establishing an artificial system still remains a challenge. Herein, abundant chirality inversion is achieved by precisely and multiply manipulating the co-assembled binding sites of phenylalanine derivatives (D/LPPF) with different naphthalene derivatives (NA, NC, NP, NF). The amide and hydroxy group of naphthalene derivatives prefer to bind with the carboxy group of LPPF, while carboxylic groups and fluoride atoms tend to bind with the amide moiety of LPPF. All these diverse interaction modes can precisely trigger helicity inversion of LPPF nanofibers. In addition, synergistically manipulating the carboxy and amide binding sites from a single LPPF molecule to simultaneously interact with different naphthalene derivatives leads to chirality regulation. Typically, varying the solvent may switch the interaction modes and the switched new interaction modes can be employed to further regulate the chirality of the LPPF nanofibers. This study may provide a novel approach to explore chirality diversity in artificial systems by regulating the intermolecular binding sites.

Kinetic Co-assembly Pathway Induced Chirality Inversion Along with Morphology Transition.

Kinetic co-assembly pathway induced chirality inversion along with morphology transition is of importance to understand biological processes, but still remains a challenge to realize in artificial systems. Herein, helical nanofibers consisting of phenylalanine-based enantiomers (L/DPF) successfully transform into kinetically trapped architectures with opposite helicity through a kinetic co-assembly pathway. By contrast, the co-assemblies obtained by a thermodynamic pathway exhibit non-helical structures. The formation sequence of non-covalent interactions plays a crucial role in structural chirality of co-assemblies. For the kinetic pathway, the hydrogen bonding between D/LPF and naphthylamide derivatives forms before π-π stacking to facilitate the formation of helical structures with inverse handedness. This study may provide an approach to explore chirality inversion accompanied by morphology transition by manipulating the kinetic co-assembly pathway.

Supramolecular Hydrogels with Tunable Chirality for Promising Biomedical Applications.

Chirality exits from molecular-level, supramolecular, and nanoscaled helical structures to the macroscopic level in biological life. Among these various levels, as the central structural motifs in living systems (e.g., double helix in DNA, α-helix, ß-sheet in proteins), supramolecular helical systems arising from the asymmetrical spatial stacking of molecular units play a crucial role in a wide diversity of biochemical reactions (e.g., gene replication, molecular recognition, ion transport, enzyme catalysis, and so on). However, the importance of supramolecular chirality and its potential biofunctions has not yet been fully explored. Thus, generating chiral assembly to transfer nature's chiral code to artificial biomaterials is expected to be utilized for developing novel functional biomaterials. As one of the most commonly used biomaterials, supramolecular hydrogels have attracted considerable research interest due to their resemblance to the structure and function of the native extracellular matrix (ECM). Therefore, the performance and manipulation of chiral assembled nanoarchitectures in supramolecular hydrogels may provide useful insights into understanding the role of supramolecular chirality in biology.In this Account, recent progress on chiral supramolecular hydrogels is presented, including how to construct and regulate assembled chiral nanostructures in hydrogels with controllable handedness and then use them to develop chiral hydrogels that could be applied in biology, biochemistry, and medicine. First, a brief introduction is provided to present the basic concept related to supramolecular chirality and the importance of supramolecular chirality in living systems. The chiral assemblies in supramolecular hydrogels are strongly driven by noncovalent interactions between molecular building blocks (such as hydrogen bonding, π-π stacking, hydrophobic, and van der Waals interactions). Consequently, the handedness of these chiral assemblies can be regulated by many extra stimuli including solvents, temperature, pH, metal ions, enzymes, and photoirradiation, which is presented in the second section. This manipulation of the chirality of nanoarchitectures in supramolecular hydrogels can result in the development of potential biofunctions. For example, specific supramolecular chirality-induced biological phenomena (such as controlled cell adhesion, proliferation, differentiation, apoptosis, protein adsorption, drug delivery, and antibacterial adhesion) are presented in detail in the third section. Finally, the outlook of open challenges and future developments of this rapidly evolving field is provided. This account that highlights the diverse chirality-dependent biological phenomena not only helps us to understand the importance of chirality in life but also provides new ideas for designing and preparing chiral materials for more bioapplications.

Rational Fabrication of Multiple Dimensional Assemblies from Tryptophan-Based Racemate.

Fabricating structural complex assemblies from simple amino acid-based derivatives is attracting great research interests due to their easy accessibility and preparation. However, the morphological regulation of racemates (an equimolar mixture of enantiomers) were largely overlooked. In this work, through rational modulation of kinetic and thermodynamic parameters, we achieved multiple dimensional architectures employing tryptophan-based racemate (RPWM). Upon assembling, 1D bundled nanofibers, 2D lamellar nanostructure and 3D urchin-like microflowers could be obtained depending on the solvents used. The corresponding morphology evolutions were successfully illustrated by changing the enantiomeric excess (ee) value. Moreover, for RPWM, uniform 0D nanospheres were formed in H2 O under 4 °C, which could spontaneously convert into lamella under ambient temperature. Taking advantages of its temperature-responsive phase change behavior, RPWM assemblies exhibited excellent removal efficiency for organic dye RhB, and could be reused for several consecutive cycles without significant changes in its removal performance. Taken together, it's rational to envision that the engineering of racemates assembly pathways can greatly increase the robustness in a wide variety of supramolecular materials and further lead to their blooming versatile applications.

Effect of Stereochemistry on Chirality and Gelation Properties of Supramolecular Self-Assemblies.

Although chiral nanostructures have been fabricated at various structural levels, the transfer and amplification of chirality from molecules to supramolecular self-assemblies are still puzzling, especially for heterochiral molecules. Herein, four series of C2 -symmetrical dipeptide-based derivatives bearing various amino acid sequences and different chiralities are designed and synthesized. The transcription and amplification of molecular chirality to supramolecular assemblies are achieved. The results show that supramolecular chirality is only determined by the amino acid adjacent to the benzene core, irrespective of the absolute configuration of the C-terminal amino acid. In addition, molecular chirality also has a significant influence on the gelation behavior. For the diphenylalanine-based gelators, the homochiral gelators can be gelled through a conventional heating-cooling process, whereas heterochiral gelators form translucent stable gels under sonication. The racemic gels possess higher mechanical properties than those of the pure enantiomers. All of these results contribute to an increasing knowledge over control of the generation of specific chiral supramolecular structures and the development of new optimized strategies to achieve functional supramolecular organogels through heterochiral and racemic systems.

Solvent-Controlled Topological Evolution from Nanospheres to Superhelices.

Supramolecular assemblies with diverse morphologies are crucial in determining their biochemical or physical properties. However, the topological evolution and self-assembly intermediates as well as the mechanism remain elusive. Herein, a dynamic morphological evolution from solid nanospheres to superhelical nanofibers is revealed via self-assembly of a minimal l-tryptophan-based derivative (LPWM) with various mixed solvent combinations, including the formation of solid nanospheres, the fusion of nanospheres into pearling necklace, the disintegration of necklace into short nanofibers, the distortion of nanofibers into nanotwists, and the entanglement of nanotwists into superhelices. It is found that the breakage of intramolecular H-bonds and reconstruction of intermolecular H-bonds, as well as the variation of aromatic interactions and hydrophobic effects, are the key driving forces for topological transformation, especially the dimensional evolution. The nanospheres and nanofibers demonstrate discrepant behaviors towards mouse neural stem cell (NSC) differentiation that compared with negligible impact of nanospheres scaffold, the nanofibers scaffold is favorable for NSC differentiation into neurons. The remarkable dynamic regulation of assembly process, together with the NSC differentiation on twisted nanofibers are making this system an ideal model to interpret complex proteins fibrillation processes and investigate the structure-function relationship.

Visible Enantiomer Discrimination via Diphenylalanine-Based Chiral Supramolecular Self-Assembly on Multiple Platforms.

The development of enantioselective recognition is of great significance in medical science and pharmaceutical industry, which associates with the molecular recognition phenomenon widely observed in biological systems. In particular, the facile and straight achievement of visual enantioselective recognition has been drawing increasing consideration, but it is still a challenge. Herein, a heterochiral diphenylalanine-based gelator (LFDF) is synthesized, presenting left-handed nanofibers during self-assembly in ethanol, which accomplishes the phenylalaninol enantiomer recognition on multiple platforms. When adding l- or d-phenylalaninol into LFDF supramolecular solution followed by ultrasonic treatment, precipitate and gel are formed, respectively. Meanwhile, LFDF supramolecular gel completely collapses in a minute after dropping l-phenylalaninol, while the gel almost remains when d-type is employed. Moreover, a fluorescent supramolecular xerogel (ThT-LFDF) is fabricated by combining the LFDF gelator with thioflavine T (ThT), which could detect l-phenylalaninol accompanying with fluorescence quenching while d-type with barely decreasing. And the ThT-LFDF xerogel system shows a good sensitivity (reaches to ppm) for the detection of l-phenylalaninol. It is found that the chirality of the assembled nanofibers, as well as amino and carboxyl of phenylalaninol, plays a critical role on the discrimination process. The multiple and visible enantioselective recognition of phenylalaninol through chiral supramolecular self-assemblies shows potential applications in the fields of medical science and pharmaceutical industry.

Three-Dimensional Microstructured Poly(vinyl alcohol) Hydrogel Platform for the Controlled Formation of Multicellular Cell Spheroids.

Three-dimensional (3D) multicellular cell spheroids (MCSs) are excellent in vitro cell models, in which, e.g., the in vivo cell-cell interaction processes are much better mimicked than in conventional two-dimensional (2D) cell layers. However, the difficulties in the generation of well-defined MCSs with controlled size severely limit their application. Herein, low-adhesive poly(vinyl alcohol) (PVA) hydrogels structured with inverted pyramid-shaped microwells were used to guide the aggregation of cells into MCSs. The cells settling down into the microwells by gravity accumulated at the central tip of the wells and then gradually grew into spheroids. The size of cell spheroids can be straightforwardly controlled by the culture time and initially seeded cell number. The MCSs generated in a parallel microarray format were further used for drug testing. Our results suggest in agreement with complementary literature data that the cell culture format plays a critical role in the cellular response to drugs, and also confirms that spheroids possess a much higher drug resistance than cells in 2D layers. This novel microstructured PVA hydrogel is expected to offer a potential platform for the facile preparation of spheroids for various applications in the biomedical field.

Isolated Reporter Bacteria in Supramolecular Hydrogel Microwell Arrays.

The combination of supramolecular hydrogels formed by low molecular weight gelator self-assembly via noncovalent interactions within a scaffold derived from polyethylene glycol (PEG) affords an interesting approach to immobilize fully functional, isolated reporter bacteria in novel microwell arrays. The PEG-based scaffold serves as a stabilizing element and provides physical support for the self-assembly of the C2-phenyl-derived gelator on the micrometer scale. Supramolecular hydrogel microwell arrays with various shapes and sizes were used to isolate single or small numbers of Escherichia coli TOP10 pTetR-LasR-pLuxR-GFP. In the presence of the autoinducer N-(3-oxododecanoyl) homoserine lactone, the entrapped E. coli in the hydrogel microwell arrays showed an increased GFP expression. The shape and size of microwell arrays did not influence the fluorescence intensity and the projected size of the bacteria markedly, while the population density of seeded bacteria affected the number of bacteria expressing GFP per well. The hydrogel microwell arrays can be further used to investigate quorum sensing, reflecting communication in inter- and intraspecies bacterial communities for biology applications in the field of biosensors. In the future, these self-assembled hydrogel microwell arrays can also be used as a substrate to detect bacteria via secreted autoinducers.

Chiral Supramolecular Hydrogel Enhanced Transdermal Delivery of Sodium Aescinate to Modulate M1 Macrophage Polarization Against Lymphedema.

Sodium aescinate (SA) shows great potential for treating lymphedema since it can regulate the expression of cytokines in M1 macrophages, however, it is commonly administered intravenously in clinical practice and often accompanied by severe toxic side effects and short metabolic cycles. Herein, SA-loaded chiral supramolecular hydrogels are prepared to prove the curative effects of SA on lymphedema and enhance its safety and transdermal transmission efficiency. In vitro studies demonstrate that SA- loaded chiral supramolecular hydrogels can modulate local immune responses by inhibiting M1 macrophage polarization. Typically, these chiral hydrogels can significantly increase the permeability of SA with good biocompatibility due to the high enantioselectivity between chiral gelators and stratum corneum and L-type hydrogels are found to have preferable drug penetration over D-type hydrogels. In vivo studies show that topical delivery of SA via chiral hydrogels results in dramatic therapeutic effects on lymphedema. Specifically, it can downregulate the level of inflammatory cytokines, reduce the development of fibrosis, and promote the regeneration of lymphatic vessels. This study initiates the use of SA for lymphedema treatment and for the creation of an effective chiral biological platform for improved topical administration.

Amino acid-based supramolecular chiral hydrogels promote osteogenesis of human dental pulp stem cells via the MAPK pathway.

Critical-size defects (CSDs) of the craniofacial bones cause aesthetic and functional complications that seriously impact the quality of life. The transplantation of human dental pulp stem cells (hDPSCs) is a promising strategy for bone tissue engineering. Chirality is commonly observed in natural biomolecules, yet its effect on stem cell differentiation is seldom studied, and little is known about the underlying mechanism. In this study, supramolecular chiral hydrogels were constructed using L/d-phenylalanine (L/D-Phe) derivatives. The results of alkaline phosphatase expression analysis, alizarin red S assay, as well as quantitative real-time polymerase chain reaction and western blot analyses suggest that right-handed D-Phe hydrogel fibers significantly promoted osteogenic differentiation of hDPSCs. A rat model of calvarial defects was created to investigate the regulation of chiral nanofibers on the osteogenic differentiation of hDPSCs in vivo. The results of the animal experiment demonstrated that the D-Phe group exhibited greater and faster bone formation on hDPSCs. The results of RNA sequencing, vinculin immunofluorescence staining, a calcium fluorescence probe assay, and western blot analysis indicated that L-Phe significantly promoted adhesion of hDPSCs, while D-Phe nanofibers enhanced osteogenic differentiation of hDPSCs by facilitating calcium entry into cells and activate the MAPK pathway. These results of chirality-dependent osteogenic differentiation offer a novel therapeutic strategy for the treatment of CSDs by optimising the differentiation of hDPSCs into chiral nanofibers.

Wettability of supramolecular nanofibers for controlled cell adhesion and proliferation.

By employing smart self-assembly of 1,4-benyldicarbonxamide-phenylalanine (C2) derived supramolecular gelators, a simple way to construct nanofibrous environments with the controllable wettability is developed. The fast cell adhesion and proliferation on the least wettable fibers indicates an efficient control over cells, which is proved to be mainly mediated by the interaction between protein and the fibers. One typical merit superior to other materials is that cell adhesion can be regulated not only on two-dimensional (2D) substrates but also in three-dimensional (3D) microenvironments. This paves a novel way to deeply understand the influence of fiber wettability on cell behaviors in 3D environment.

Dual-specific interaction to detect DNA on gold nanoparticles.

An approach to selectively and efficiently detect single strand DNA is developed by using streptavidin coated gold nanoparticles (StAuNPs) as efficient quenchers. The central concept for the successful detection is the combination the of streptavidin-biotin interaction with specific probe-target DNA hybridization. Biotin labeled probe DNAs act as "bridges" to bring Cy5 labeled targets to the particle surface and the fluorophore dye can be rapidly and efficiently quenched by StAuPNs. By measuring the changes of photoluminescence intensity of Cy5, an efficient, selective, and reversed detection of DNA hybridization is realized. The methodology may pave a new way for simple and rapid detections of biomolecules.

Chiral Supramolecular Assemblies: Controllable Construction and Biological Activity.

Chiral supramolecular assemblies with helical structures (e. g., proteins with α-helix, DNA with double helix, collagen with triple-helix) as the central structure motifs in biological systems play a crucial role in various physiological activities of living organisms. Variations in chiral structure can cause many abnormal physiological activities. To gain insight into the construction, structural transition, and related physiological functions of these complex helix in natural systems, it is necessary to fabricate artificial supramolecular assemblies with controllable helix orientation as research platform. This review discusses recent advances in chiral supramolecular assembly, including the precise construction and regulation of assembled chiral nanostructures with tunable chirality. Chiral structure-dependent biological activities, including cell proliferation, cell differentiation, antibacterial activity and tissue regeneration, are also discussed. This review not only contributes to further understanding of the importance of chirality in the physiological environment, but also plays an important role in the development of chiral biomedical materials for the treatment of diseases (e. g., tissue engineering regeneration, stem cell transplantation therapy).

Infected Diabetic Wound Regeneration Using Peptide-Modified Chiral Dressing to Target Revascularization.

Revascularization plays a critical role in the healing of diabetic wounds. Accumulation of advanced glycation end products (AGEs) and refractory multidrug resistant bacterial infection are the two major barriers to revascularization, directly leading to impaired healing of diabetic wounds. Here, an artfully designed chiral gel dressing is fabricated (named as HA-LM2-RMR), which consists of l-phenylalanine and cationic hexapeptide coassembled helical nanofibers cross-linked with hyaluronic acid via hydrogen bonding. This chiral gel possesses abundant chiral and cationic sites, not only effectively reducing AGEs via stereoselective interaction but also specifically killing multidrug resistant bacteria rather than host cells since cationic hexapeptides selectively interact with negatively charged microbial membrane. Surprisingly, the HA-LM2-RMR fibers present an attractive ability to activate sprouted angiogenesis of Human Umbilical Vein Endothelial Cells by upregulating VEGF and OPA1 expression. In comparison with clinical Prontosan Wound Gel, the HA-LM2-RMR gel presents superior healing efficiency in the infected diabetic wound with respect to angiogenesis and re-epithelialization, shortening the healing period from 21 days to 14 days. These findings for chiral wound dressing provide insights for the design and construction of diabetic wound dressings that target revascularization, which holds great potential to be utilized in tissue regenerative medicine.

Unique Chirality Selection in Neural Cells for D-Matrix Enabling Specific Manipulation of Cell Behaviors.

Manipulating neural cell behaviors is a critical issue to various therapies for neurological diseases and damages, where matrix chirality has long been overlooked despite the proven adhesion and proliferation improvement of multiple non-neural cells by L-matrixes. Here, it is reported that the D-matrix chirality specifically enhances cell density, viability, proliferation, and survival in four different types of neural cells, contrasting its inhibition in non-neural cells. This universal impact on neural cells is defined as "chirality selection for D-matrix" and is achieved through the activation of JNK and p38/MAPK signaling pathways by the cellular tension relaxation resulting from the weak interaction between D-matrix and cytoskeleton proteins, particularly actin. Also, D-matrix promotes sciatic nerve repair effectively, both with or without non-neural stem cell implantation, by improving the population, function, and myelination of autologous Schwann cells. D-matrix chirality, as a simple, safe, and effective microenvironment cue to specifically and universally manipulate neural cell behaviors, holds extensive application potential in addressing neurological issues such as nerve regeneration, neurodegenerative disease treatment, neural tumor targeting, and neurodevelopment.

A highly efficient self-assembly of responsive C(2) -cyclohexane-derived gelators.

The rational design and synthesis of a family of effective low-molecular-weight gelators (LMWGs) with a modular architecture based on a C(2) -1,4-diamide cyclohexane core are reported. Due to the high symmetry, the gelators are initially well distributed in solution and no trapped aggregates are formed before the assembly is triggered. The subsequent self-assembly process, which results in the formation of versatile gels, is highly efficient and can be triggered and tuned due to its remarkable dependence on the pH of solution. The assembly of different gelators is found to be closely related to the pK(a) of the corresponding functional substituents on the LMWGs. Primary cell culture experiments reveal that the hydrogels made under physiological conditions are promising as potential tailor-made microenvironments.