Templated Self‐Assembly of Block Copolymers: Top‐Down Helps Bottom‐Up

One of the key challenges in nanotechnology is to control a self‐assembling system to create a specific structure. Self‐organizing block copolymers offer a rich variety of periodic nanoscale patterns, and researchers have succeeded in finding conditions that lead to very long range order of the domains. However, the array of microdomains typically still contains some uncontrolled defects and lacks global registration and orientation. Recent efforts in templated self‐assembly of block copolymers have demonstrated a promising route to control bottom‐up self‐organization processes through top‐down lithographic templates. The orientation and placement of block‐copolymer domains can be directed by topographically or chemically patterned templates. This templated self‐assembly method provides a path towards the rational design of hierarchical device structures with periodic features that cover several length scales.     

Dynamic Self‐Assembly of Homogenous Microcyclic Structures Controlled by a Silver‐Coated Nanopore

The self‐assembly of nanoparticles is a challenging process for organizing precise structures with complicated and ingenious structures. In the past decades, a simple, high‐efficiency, and reproducible self‐assembly method from nanoscale to microscale has been pursued because of the promising and extensive application prospects in bioanalysis, catalysis, photonics, and energy storage. However, microscale self‐assembly still faces big challenges including improving the stability and homogeneity as well as pursuing new assembly methods and templates for the uniform self‐assembly. To address these obstacles, here, a novel silver‐coated nanopore is developed which serves as a template for electrochemically generating microcyclic structures of gold nanoparticles at micrometers with highly homogenous size and remarkable reproducibility. Nanopore‐induced microcyclic structures are further applied to visualize the diffusion profile of ionic flux. Based on this novel strategy, a nanopore could potentially facilitate the delivery of assembled structures for many practical applications including drug delivery, cellular detection, catalysis, and plasmonic sensing.     

Multiscale Experimental Mechanics of Hierarchical Carbon‐Based Materials

Investigation of the mechanics of natural materials, such as spider silk, abalone shells, and bone, has provided great insight into the design of materials that can simultaneously achieve high specific strength and toughness. Research has shown that their emergent mechanical properties are owed in part to their specific self‐organization in hierarchical molecular structures, from nanoscale to macroscale, as well as their mixing and bonding. To apply these findings to manmade materials, researchers have devoted significant efforts in developing a fundamental understanding of multiscale mechanics of materials and its application to the design of novel materials with superior mechanical performance. These efforts included the utilization of some of the most promising carbon‐based nanomaterials, such as carbon nanotubes, carbon nanofibers, and graphene, together with a variety of matrix materials. At the core of these efforts lies the need to characterize material mechanical behavior across multiple length scales starting from nanoscale characterization of constituents and their interactions to emerging micro‐ and macroscale properties. In this report, progress made in experimental tools and methods currently used for material characterization across multiple length scales is reviewed, as well as a discussion of how they have impacted our current understanding of the mechanics of hierarchical carbon‐based materials. In addition, insight is provided into strategies for bridging experiments across length scales, which are essential in establishing a multiscale characterization approach. While the focus of this progress report is in experimental methods, their concerted use with theoretical‐computational approaches towards the establishment of a robust material by design methodology is also discussed, which can pave the way for the development of novel materials possessing unprecedented mechanical properties.     

Dual‐Phase Separation in a Semiconfined System: Monodispersed Heterogeneous Block‐Copolymer Membranes for Cell Encoding and Patterning

Block copolymers (BCPs) have the capacity to self‐assemble into a myriad of well‐defined aggregate structures, offering great promise for the construction of drug delivery, photolithographic templates, and complex nanoscale assemblies. A uniqueness of these materials is their propensity to become kinetically frozen in non‐equilibrium states, implying that the process of self‐assembly can be utilized to remodel the resulting structures. Here, a new semiconfined system for processing the BCP self‐assembly is constructed, in which an unusual dual‐phase separation occurs, including nonsolvent‐induced microphase separation and osmotically driven macrophase separation, ultimately yielding heterogeneous BCP membranes. These membranes with cellular dimensions show unique anisotropy that can be used for cell encoding and patterning, which are highly relevant to biology and medicine. This processing method not only provides new levels of tailorability to the structures and encapsulated contents of BCP assemblies, but can also be generalized to other block polymers, particularly those with attractive electronic and/or optical properties.     

Self‐Powered Intracellular Drug Delivery by a Biomechanical Energy‐Driven Triboelectric Nanogenerator

Nondestructive, high‐efficiency, and on‐demand intracellular drug/biomacromolecule delivery for therapeutic purposes remains a great challenge. Herein, a biomechanical‐energy‐powered triboelectric nanogenerator (TENG)‐driven electroporation system is developed for intracellular drug delivery with high efficiency and minimal cell damage in vitro and in vivo. In the integrated system, a self‐powered TENG as a stable voltage pulse source triggers the increase of plasma membrane potential and membrane permeability. Cooperatively, the silicon nanoneedle‐array electrode minimizes cellular damage during electroporation via enhancing the localized electrical field at the nanoneedle–cell interface and also decreases plasma membrane fluidity for the enhancement of molecular influx. The integrated system achieves efficient delivery of exogenous materials (small molecules, macromolecules, and siRNA) into different types of cells, including hard‐to‐transfect primary cells, with delivery efficiency up to 90% and cell viability over 94%. Through simple finger friction or hand slapping of the wearable TENGs, it successfully realizes a transdermal biomolecule delivery with an over threefold depth enhancement in mice. This integrated and self‐powered system for active electroporation drug delivery shows great prospect for self‐tuning drug delivery and wearable medicine.     

Anisotropic Crystalline Protein Nanolayers as Multi‐Functional Biointerface for Patterned Co‐Cultures of Adherent and Non‐Adherent Cells in Microfluidic Devices

The spatial arrangement of cells in their microenvironment is known to significantly influence cellular behavior, thus making the control of cellular organization an important parameter of in vitro co‐culture models. However, recent advances in micropatterning co‐culture methods within biochips do not address the simultaneous cultivation of anchorage‐dependent and non‐adherent cells. To address this methodological gap we combine S‐layer technology with microfluidics to pattern co‐cultures to study the cell‐to‐cell and cell‐to‐surface interactions under physiologically relevant conditions. We exploit the unique self‐assembly properties of SbpA and SbsB S‐layers to create an anisotropic protein nanobiointerface on‐chip with spatially‐defined cytophilic (adhesive) and cytophobic (repulsive) properties. While microfluidics control physical parameters such as shear force and flow velocities, our anisotropic protein nanobiointerface regulates the biological aspects of the co‐culture method including biocompatibility, biostability, and affinity to non‐adherent cells. The reliability and reproducibility of our microfluidic co‐culture strategy based on laminar flow patterned protein nanolayers is envisioned to advance in vitro models for biomedical research.     

Mixed Fluorinated/Hydrogenated Self‐Assembled Monolayer‐Protected Gold Nanoparticles: In Silico and In Vitro Behavior

Gold nanoparticles (AuNPs) covered with mixtures of immiscible ligands present potentially anisotropic surfaces that can modulate their interactions at complex nano–bio interfaces. Mixed, self‐assembled, monolayer (SAM)‐protected AuNPs, prepared with incompatible hydrocarbon and fluorocarbon amphiphilic ligands, are used here to probe the molecular basis of surface phase separation and disclose the role of fluorinated ligands on the interaction with lipid model membranes and cells, by integrating in silico and experimental approaches. These results indicate that the presence of fluorinated amphiphilic ligands enhances the membrane binding ability and cellular uptake of gold nanoparticles with respect to those coated only with hydrogenated amphiphilic ligands. For mixed monolayers, computational results suggest that ligand phase separation occurs on the gold surface, and the resulting anisotropy affects the number of contacts and adhesion energies with a membrane bilayer. This reflects in a diverse membrane interaction for NPs with different surface morphologies, as determined by surface plasmon resonance, as well as differential effects on cells, as observed by flow cytometry and confocal microscopy. Overall, limited changes in monolayer features can significantly affect NP surface interfacial properties, which, in turn, affect the interaction of SAM‐AuNPs with cellular membranes and subsequent effects on cells.     

Flexible Fabrication of Shape‐Controlled Collagen Building Blocks for Self‐Assembly of 3D Microtissues

Creating artificial tissue‐like structures that possess the functionality, specificity, and architecture of native tissues remains a big challenge. A new and straightforward strategy for generating shape‐controlled collagen building blocks with a well‐defined architecture is presented, which can be used for self‐assembly of complex 3D microtissues. Collagen blocks with tunable geometries are controllably produced and released via a membrane‐templated microdevice. The formation of functional microtissues by embedding tissue‐specific cells into collagen blocks with expression of specific proteins is described. The spontaneous self‐assembly of cell‐laden collagen blocks into organized tissue constructs with predetermined configurations is demonstrated, which are largely driven by the synergistic effects of cell–cell and cell–matrix interactions. This new strategy would open up new avenues for the study of tissue/organ morphogenesis, and tissue engineering applications.     

Colloidal Self‐Assembly Meets Nanofabrication: From Two‐Dimensional Colloidal Crystals to Nanostructure Arrays

Self‐assembly of colloidal microspheres or nanospheres is an effective strategy for fabrication of ordered nanostructures. By combination of colloidal self‐assembly with nanofabrication techniques, two‐dimensional (2D) colloidal crystals have been employed as masks or templates for evaporation, deposition, etching, and imprinting, etc. These methods are defined as “colloidal lithography”, which is now recognized as a facile, inexpensive, and repeatable nanofabrication technique. This paper presents an overview of 2D colloidal crystals and nanostructure arrays fabricated by colloidal lithography. First, different methods for fabricating self‐assembled 2D colloidal crystals and complex 2D colloidal crystal structures are summarized. After that, according to the nanofabrication strategy employed in colloidal lithography, related works are reviewed as colloidal‐crystal‐assisted evaporation, deposition, etching, imprinting, and dewetting, respectively.     

Three‐Dimensional Fabrication at Small Size Scales

Despite the fact that we live in a 3D world and macroscale engineering is 3D, conventional submillimeter‐scale engineering is inherently 2D. New fabrication and patterning strategies are needed to enable truly 3D‐engineered structures at small size scales. Here, strategies that have been developed over the past two decades that seek to enable such millimeter to nanoscale 3D fabrication and patterning are reviewed. A focus is the strategy of self‐assembly, specifically in a biologically inspired, more deterministic form, known as self‐folding. Self‐folding methods can leverage the strengths of lithography to enable the construction of precisely patterned 3D structures and “smart” components. This self‐assembly approach is compared with other 3D fabrication paradigms, and its advantages and disadvantages are discussed.

     

Interface‐Directed Self‐Assembly of Cell‐Laden Microgels

Cell‐laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer‐scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter‐scale, cell‐laden hydrogels through the assembly of shape‐controlled microgels or a liquid–air interface. Cell‐laden microgels of specific shapes are randomly placed on the surface of a high‐density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue‐like structures. The resulting assemblies are cell‐laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air–liquid interface can be used to self‐assemble spatially controllable, cocultured tissue‐like structures.     

Molecular Self‐Assembly on Graphene

The formation of ordered arrays of molecules via self‐assembly is a rapid, scalable route towards the realization of nanoscale architectures with tailored properties. In recent years, graphene has emerged as an appealing substrate for molecular self‐assembly in two dimensions. Here, the first five years of progress in supramolecular organization on graphene are reviewed. The self‐assembly process can vary depending on the type of graphene employed: epitaxial graphene, grown in situ on a metal surface, and non‐epitaxial graphene, transferred onto an arbitrary substrate, can have different effects on the final structure. On epitaxial graphene, the process is sensitive to the interaction between the graphene and the substrate on which it is grown. In the case of graphene that strongly interacts with its substrate, such as graphene/Ru(0001), the inhomogeneous adsorption landscape of the graphene moiré superlattice provides a unique opportunity for guiding molecular organization, since molecules experience spatially constrained diffusion and adsorption. On weaker‐interacting epitaxial graphene films, and on non‐epitaxial graphene transferred onto a host substrate, self‐assembly leads to films similar to those obtained on graphite surfaces. The efficacy of a graphene layer for facilitating planar adsorption of aromatic molecules has been repeatedly demonstrated, indicating that it can be used to direct molecular adsorption, and therefore carrier transport, in a certain orientation, and suggesting that the use of transferred graphene may allow for predictible molecular self‐assembly on a wide range of surfaces.     

Autonomous Ultrafast Self‐Healing Hydrogels by pH‐Responsive Functional Nanofiber Gelators as Cell Matrices

The synthesis of hybrid hydrogels by pH‐controlled structural transition with exceptional rheological properties as cellular matrix is reported. “Depsi” peptide sequences are grafted onto a polypeptide backbone that undergo a pH‐induced intramolecular O–N–acyl migration at physiological conditions affording peptide nanofibers (PNFs) as supramolecular gelators. The polypeptide–PNF hydrogels are mechanically remarkably robust. They reveal exciting thixotropic behavior with immediate in situ recovery after exposure to various high strains over long periods and self‐repair of defects by instantaneous reassembly. High cytocompatibility, convenient functionalization by coassembly, and controlled enzymatic degradation but stability in 2D and 3D cell culture as demonstrated by the encapsulation of primary human umbilical vein endothelial cells and neuronal cells open many attractive opportunities for 3D tissue engineering and other biomedical applications.     

4D Biofabrication Using Shape‐Morphing Hydrogels

Despite the tremendous potential of bioprinting techniques toward the fabrication of highly complex biological structures and the flourishing progress in 3D bioprinting, the most critical challenge of the current approaches is the printing of hollow tubular structures. In this work, an advanced 4D biofabrication approach, based on printing of shape‐morphing biopolymer hydrogels, is developed for the fabrication of hollow self‐folding tubes with unprecedented control over their diameters and architectures at high resolution. The versatility of the approach is demonstrated by employing two different biopolymers (alginate and hyaluronic acid) and mouse bone marrow stromal cells. Harnessing the printing and postprinting parameters allows attaining average internal tube diameters as low as 20 µm, which is not yet achievable by other existing bioprinting/biofabrication approaches and is comparable to the diameters of the smallest blood vessels. The proposed 4D biofabrication process does not pose any negative effect on the viability of the printed cells, and the self‐folded hydrogel‐based tubes support cell survival for at least 7 d without any decrease in cell viability. Consequently, the presented 4D biofabrication strategy allows the production of dynamically reconfigurable architectures with tunable functionality and responsiveness, governed by the selection of suitable materials and cells.     

Photoresponsive Cucurbit[8]uril‐Mediated Adhesion of Bacteria on Supported Lipid Bilayers

In this work, the development of a photoresponsive platform for the presentation of bioactive ligands to study receptor–ligand interactions has been described. For this purpose, supramolecular host–guest chemistry and supported lipid bilayers (SLBs) have been combined in a microfluidic device. Quartz crystal microbalance with dissipation monitoring (QCM‐D) studies on methyl viologen (MV)‐functionalized oligo ethylene glycol‐based self‐assembled monolayers, gel and liquid‐state SLBs have been compared for their nonfouling properties in the case of ConA and bacteria. In combination with bacterial adhesion test, negligible nonspecific bacterial adhesion is observed only in the case of methyl‐viologen‐modified liquid‐state SLBs. Therefore, liquid‐state SLBs have been identified as most suitable for studying specific cell interactions when MV is incorporated as a guest on the surface. The photoswitchable supramolecular ternary complex is formed by assembling cucurbit[8]uril (CB[8]) and an azobenzene–mannose conjugate (Azo–Man) onto MV‐functionalized liquid‐state SLBs and the assembly process has been characterized using QCM‐D and fluorescence techniques. Mannose has been found to enable binding of E. coli via cell‐surface receptors on the nonfouling supramolecular SLBs. Optical switching of the azobenzene moiety allows us to “erase” the bioactive surface after bacterial binding, providing the potential to develop reusable sensors. Localized photorelease of bacterial cells has also been shown indicating the possibility of optically guiding cellular growth, migration, and intercellular interactions.     

One-Step Rapid Fabrication of Cell-Only Living Fibers

Cellular aggregates are used as relevant regenerative building blocks, tissue models, and cell delivery platforms. Biomaterial-free structures are often assembled either as 2D cell sheets or spherical microaggregates, both incompatible with free-form deposition, and dependent on challenging processes for macroscale 3D upscaling. The continuous and elongated nature of fiber-shaped materials enables their deposition in unrestricted multiple directions. Cellular fiber fabrication has often required exogenously provided support proteins and/or the use of biomaterial-based sacrificial templates. Here, the rapid (<24 h) assembly of fiberoids is reported: living centimeter-long scaffold-free fibers of cells produced in the absence of exogenous materials or supplements. Adipose-derived mesenchymal stem cell fiberoids can be easily modulated into complex multidimensional geometries and show tissue-invasive properties while keeping the secretion of trophic factors. Proangiogenic properties studied on a chick chorioallantoic membrane in an ovo model are observed for heterotypic fiberoids containing endothelial cells. These micro-to-macrotissues may find application as morphogenic therapeutic and tissue-mimetic building blocks, with the ability to integrate 3D and 4D full biological materials.     

Rational Design of Membrane‐Pore‐Forming Peptides

Specific interactions of peptides with lipid membranes are essential for cellular communication and constitute a central aspect of the innate host defense against pathogens. A computational method for generating innovative membrane‐pore‐forming peptides inspired by natural templates is presented. Peptide representation in terms of sequence‐ and topology‐dependent hydrophobic moments is introduced. This design concept proves to be appropriate for the de novo generation of first‐in‐class membrane‐active peptides with the anticipated mode of action. The designed peptides outperform the natural template in terms of their antibacterial activity. They form a kinked helical structure and self‐assemble in the membrane by an entropy‐driven mechanism to form dynamically growing pores that are dependent on the lipid composition. The results of this study demonstrate the unique potential of natural template‐based peptide design for chemical biology and medicinal chemistry.     

A Self‐Assembled DNA Origami‐Gold Nanorod Complex for Cancer Theranostics

A self‐assembled DNA origami (DO)‐gold nanorod (GNR) complex, which is a dual‐functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO‐GNR complexes with human MCF7 breast cancer cells, significant enhancement of cell uptake is achieved compared to bare GNRs by two‐photon luminescence imaging. Particularly, the triangle shaped DO‐GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO‐GNR complex by two‐photon laser or NIR laser irradiation. Moreover, the DO‐GNR complex exhibits enhanced antitumor efficacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO‐GNR complex can achieve optimal two‐photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo.     

Enzyme‐Instructed Supramolecular Self‐Assembly with Anticancer Activity

Cancer remains one of the leading causes of death, which has continuously stimulated the development of numerous functional biomaterials with anticancer activities. Herein is reviewed one recent trend of biomaterials focusing on the advances in enzyme‐instructed supramolecular self‐assembly (EISA) with anticancer activity. EISA relies on enzymatic transformations to convert designed small‐molecular precursors into corresponding amphiphilic residues that can form assemblies in living systems. EISA has shown some advantages in controlling cell fate from three aspects. 1) Based on the abnormal activity of specific enzymes, EISA can differentiate cancer cells from normal cells. In contrast to the classical ligand–receptor recognition, the targeting capability of EISA relies on dynamic control of the self‐assembly process. 2) The interactions between EISA and cellular components directly disrupt cellular processes or pathways, resulting in cell death phenotypes. 3) EISA spatiotemporally controls the distribution of therapeutic agents, which boosts drug delivery efficiency. Therefore, with regard to the development of EISA, the aim is to provide a perspective on the future directions of research into EISA as anticancer theranostics.     

3D Mimicry of Native‐Tissue‐Fiber Architecture Guides Tendon‐Derived Cells and Adipose Stem Cells into Artificial Tendon Constructs

Tendon and ligament (T/L) function is intrinsically related with their unique hierarchically and anisotropically organized extracellular matrix. Their natural healing capacity is, however, limited. Here, continuous and aligned electrospun nanofiber threads (CANT) based on synthetic/natural polymer blends mechanically reinforced with cellulose nanocrystals are produced to replicate the nanoscale collagen fibrils grouped into microscale collagen fibers that compose the native T/L. CANT are then incrementally assembled into 3D hierarchical scaffolds, resulting in woven constructions, which simultaneously mimic T/L nano‐to‐macro architecture, nanotopography, and nonlinear biomechanical behavior. Biological performance is assessed using human‐tendon‐derived cells (hTDCs) and human adipose stem cells (hASCs). Scaffolds nanotopography and microstructure induce a high cytoskeleton elongation and anisotropic organization typical of tendon tissues. Moreover, the expression of tendon‐related markers (Collagen types I and III, Tenascin‐C, and Scleraxis) by both cell types, and the similarities observed on their expression patterns over time suggest that the developed scaffolds not only prevent the phenotypic drift of hTDCs, but also trigger tenogenic differentiation of hASCs. Overall, these results demonstrate a feasible approach for the scalable production of 3D hierarchical scaffolds that exhibit key structural and biomechanical properties, which can be advantageously explored in acellular and cellular T/L TE strategies.