Design of Multistimuli Responsive Hydrogels Using Integrated Modeling and Genetically Engineered Silk–Elastin‐Like Proteins

Elastomeric, robust, and biocompatible hydrogels are rare, while the need for these types of biomaterials in biomedical‐related uses remains high. Here, a new family of genetically engineered silk–elastin‐like proteins (SELPs) with encoded enzymatic crosslinking sites is developed for a new generation of stimuli‐responsive yet robust hydrogels. Input into the designs is guided by simulation and realized via genetic engineering strategies. The avoidance of gamma irradiation or chemical crosslinking during gel fabrication, in lieu of an enzymatic process, expands the versatility of these new gels for the incorporation of labile proteins and cells. In the present study, the new SELP hydrogels offer sequence‐dependent, reversible stimuli‐responsive features. Their stiffness covers almost the full range of the elasticity of soft tissues. Further, physical modification of the silk domains provides a secondary control point to fine‐tune mechanical stiffness while preserving stimuli‐responsive features, with implications for a variety of biomedical material and device needs.     

A Highly Elastic and Rapidly Crosslinkable Elastin‐Like Polypeptide‐Based Hydrogel for Biomedical Applications

Elastin‐like polypeptides (ELPs) are promising for biomedical applications due to their unique thermoresponsive and elastic properties. ELP‐based hydrogels have been produced through chemical and enzymatic crosslinking or photocrosslinking of modified ELPs. Herein, a photocrosslinked ELP gel using only canonical amino acids is presented. The inclusion of thiols from a pair of cysteine residues in the ELP sequence allows disulfide bond formation upon exposure to UV light, leading to the formation of a highly elastic hydrogel. The physical properties of the resulting hydrogel such as mechanical properties and swelling behavior can be easily tuned by controlling ELP concentrations. The biocompatibility of the engineered ELP hydrogels is shown in vitro as well as corroborated in vivo with subcutaneous implantation of hydrogels in rats. ELP constructs demonstrate long‐term structural stability in vivo, and early and progressive host integration with no immune response, suggesting their potential for supporting wound repair. Ultimately, functionalized ELPs demonstrate the ability to function as an in vivo hemostatic material over bleeding wounds.     

High‐Throughput Topographic,Mechanical, and Biological Screening of Multilayer Films Containing Mussel‐Inspired Biopolymers

A high‐content screening method to characterize multifunctional multilayer films that combine mechanical adhesion and favorable biological response is reported. Distinct combinations of nanostructured films are produced using layer‐by‐layer methodology and their morphological, physicochemical, and biological properties are analyzed in a single microarray chip. Inspired by the composition of the adhesive proteins in mussels, thin films containing dopamine‐modified hyaluronic acid are studied. Flat biomimetic superhydrophobic patterned chips produced by a bench‐top methodology are used for the build‐up of arrays of multilayer films. The wettability contrasts imprinted onto the chips are allowed to produce individual, position controlled, multilayer films in the wettable regions. The flat configuration of the chip permits to perform a series of nondestructive measurements directly on the individual spots. In situ adhesion properties are directly measured in each spot, showing that nanostructured films richer in dopamine promote the adhesion. In vitro tests show an enhanced cell adhesion for the films with more catechol groups. The advantages presented by this platform include ability to control the uniformity and size of the multilayers films, its suitability to be used as a new low cost toolbox and for high‐content cellular screening, and capability for monitoring in situ a variety of distinct material properties.     

High Throughput,Polymeric Aqueous Two‐Phase Printing of Tumor Spheroids

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti‐cancer drugs. Two immiscible polymeric aqueous solutions are used and a submicroliter drop of the “patterning” phase containing cells is microprinted into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well‐defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, a phase diagram is established to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long‐term incubation and dose‐dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically relevant 3D tumor models.     

Covalently Adaptable Elastin‐Like Protein–Hyaluronic Acid (ELP–HA) Hybrid Hydrogels with Secondary Thermoresponsive Crosslinking for Injectable Stem Cell Delivery

Shear‐thinning, self‐healing hydrogels are promising vehicles for therapeutic cargo delivery due to their ability to be injected using minimally invasive surgical procedures. An injectable hydrogel using a novel combination of dynamic covalent crosslinking with thermoresponsive engineered proteins is presented. Ex situ at room temperature, rapid gelation occurs through dynamic covalent hydrazone bonds by simply mixing two components: hydrazine‐modified elastin‐like protein (ELP) and aldehyde‐modified hyaluronic acid. This hydrogel provides significant mechanical protection to encapsulated human mesenchymal stem cells during syringe needle injection and rapidly recovers after injection to retain the cells homogeneously within a 3D environment. In situ, the ELP undergoes a thermal phase transition, as confirmed by coherent anti‐Stokes Raman scattering microscopy observation of dense ELP thermal aggregates. The formation of the secondary network reinforces the hydrogel and results in a tenfold slower erosion rate compared to a control hydrogel without secondary thermal crosslinking. This improved structural integrity enables cell culture for three weeks postinjection, and encapsulated cells maintain their ability to differentiate into multiple lineages, including chondrogenic, adipogenic, and osteogenic cell types. Together, these data demonstrate the promising potential of ELP–HA hydrogels for injectable stem cell transplantation and tissue regeneration.     

Bio‐Orthogonally Crosslinked,Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation

Covalently‐crosslinked hydrogels are commonly used as 3D matrices for cell culture and transplantation. However, the crosslinking chemistries used to prepare these gels generally cross‐react with functional groups present on the cell surface, potentially leading to cytotoxicity and other undesired effects. Bio‐orthogonal chemistries have been developed that do not react with biologically relevant functional groups, thereby preventing these undesirable side reactions. However, previously developed biomaterials using these chemistries still possess less than ideal properties for cell encapsulation, such as slow gelation kinetics and limited tuning of matrix mechanics and biochemistry. Here, engineered elastin‐like proteins (ELPs) are developed that crosslink via strain‐promoted azide‐alkyne cycloaddition (SPAAC) or Staudinger ligation. The SPAAC‐crosslinked materials form gels within seconds and complete gelation within minutes. These hydrogels support the encapsulation and phenotypic maintenance of human mesenchymal stem cells, human umbilical vein endothelial cells, and murine neural progenitor cells. SPAAC‐ELP gels exhibit independent tuning of stiffness and cell adhesion, with significantly improved cell viability and spreading observed in materials containing a fibronectin‐derived arginine‐glycine‐aspartic acid (RGD) domain. The crosslinking chemistry used permits further material functionalization, even in the presence of cells and serum. These hydrogels are anticipated to be useful in a wide range of applications, including therapeutic cell delivery and bioprinting.     

Screening of Nanocomposite Scaffolds Arrays Using Superhydrophobic‐Wettable Micropatterns

Platforms containing multiple arrays for high‐throughput screening are demanded in the development of biomaterial libraries. Here, an array platform for the combinatorial analysis of cellular interactions and 3D porous biomaterials is described. Using a novel method based on computer‐aided manufacturing, wettable regions are printed on superhydrophobic surfaces, generating isolated spots. This freestanding benchtop array is used as a tool to deposit naturally derived polymers, chitosan and hyaluronic acid, with bioactive glass nanoparticles (BGNPs) to obtain a scaffold matrix. The effect of fibronectin adsorption on the scaffolds is also tested. The biomimetic nanocomposite scaffolds are shown to be osteoconductive, non‐cytotoxic, promote cell adhesion, and regulate osteogenic commitment. The method proves to be suitable for screening of biomaterials in 3D cell cultures as it can recreate a multitude of combinations on a single platform and identify the optimal composition that drives to desired cell responses. The platforms are fully compatible with commercially routine cell culture labware and established characterization methods, allowing for a standard control and easy adaptability to the cell culture environment. This study shows the value of 3D structured array platforms to decode the combinatorial interactions at play in cell microenvironments.     

High‐Throughput Combinatorial Optimizations of Perovskite Light‐Emitting Diodes Based on All‐Vacuum Deposition

Light‐emitting diodes (LEDs) based on lead halide perovskites demonstrate outstanding optoelectronic properties and are strong competitors for display and lighting applications. While previous halide perovskite LEDs are mainly produced via solution processing, here an all‐vacuum processing method is employed to construct CsPbBr₃ LEDs because vacuum processing exhibits high reliability and easy integration with existing OLED facilities for mass production. The high‐throughput combinatorial strategies are further adopted to study perovskite composition, annealing temperature, and functional layer thickness, thus significantly speeding up the optimization process. The best rigid device shows a current efficiency (CE) of 4.8 cd A^?1 (EQE of 1.45%) at 2358 cd m^?2, and best flexible device shows a CE of 4.16 cd A^?1 (EQE of 1.37%) at 2012 cd m^?2 with good bending tolerance. Moreover, by choosing NiO_x as the hole‐injection layer, the CE is improved to 10.15 cd A^?1 and EQE is improved to a record of 3.26% for perovskite LEDs produced by vacuum deposition. The time efficient combinatorial approaches can also be applied to optimize other perovskite LEDs.     

Understanding the Mechanical Properties of Antheraea Pernyi Silk—From Primary Structure to Condensed Structure of the Protein

Antheraea pernyi (A. pernyi) silk is produced and used by “wild” silkworms to construct a cocoon, but the primary structure of its protein is rather similar to that of spider major ampullate silk used to build web and dragline. Studies on this specific silk may provide valuable knowledge about the structure‐property relationship for the whole animal silk family. In this work, A. pernyi silk fibers with few macroscale defects are obtained by forcibly reeling, and are investigated in detail. It is found that such silk fibers display breaking stress and toughness of the same magnitude as spider major ampullate silks and forcibly reeled mulberry silk. The other mechanical properties, such as elasticity, supercontraction, and the effect of water on modulus are between those of spider major ampullate silks and mulberry silk. Therefore, an interpretation of the connection between the primary structures of silk proteins and the mechanical properties of silks is proposed here based on the ordered fraction, which in turn is determined by both the protein sequence and spinning process of the silk.     

Accelerating the Screening of Perovskite Compositions for Photovoltaic Applications through High‐Throughput Inkjet Printing

The exploration and optimization of numerous mixed perovskite compositions are causing a strong demand for high‐throughput synthesis. Nevertheless high‐throughput fabrication of perovskite films with representative film properties, which can efficiently screen the perovskite compositions for photovoltaic applications, has rarely been explored. A high‐throughput inkjet printing approach that can automatically fabricate perovskite films with various compositions with high reproducibility and high speed is developed. The automatic sequential printing of four precursors forms 25 mixed films in a fast and reproducible manner. The obtained bandgaps, photoluminescence (PL) peak positions, and PL lifetimes allow for the efficient screening of perovskite compositions for photovoltaic applications. To exemplify this concept, among 25 tested films, two compositions CH₃NH₃PbBr_0.75I_2.25 (MA) and (HC(NH₂)₂)_0.75(CH₃NH₃)_0.25PbBr_0.75I_2.25 (FA_0.75MA_0.25) with a long (237 ns) and short (49.0 ns) PL lifetime, respectively, are screened out for device investigations. As expected, the MA‐based device exhibits a much higher efficiency (19.0%) than that (15.3%) of the FA_0.75MA_0.25 counterpart. This efficiency improvement is mainly ascribed to a smaller dark saturate current density, a lower level of energetic disorder, more efficient charge transfer and decreased charge recombination losses, which are consistent with the much longer PL lifetime in the database.     

Scalable High‐Throughput Production of Modular Microgels for In Situ Assembly of Microporous Tissue Scaffolds

Hydrogel scaffolds that template the regeneration of tissue structures are widely explored; however, there is often a trade‐off between material properties, such as stiffness and interconnected pore size, that may be equally important in supporting tissue growth. Microporous annealed particle scaffolds are introduced to address this trade‐off while maintaining a flowable precursor; however, manufacturing throughput, reproducibility, and flexibility of hydrogel microparticle building blocks are limited, hindering widespread adoption. The scalable high‐throughput production of bioactive microgels for the formation of microporous tissue scaffolds in situ is presented. Using a parallelized step emulsification device, scalable high‐throughput generation of monodisperse microgels is achieved. Crosslinking is initiated downstream of droplet generation using pH modulation via proton acceptors dissolved in the oil phase. This approach enables continuous production of microgels for over 12 h while ensuring highly uniform physicochemical properties. Using this platform, the effects of local matrix stiffness on cell growth orthogonal to scaffold porosity are studied. Formation of injectable cell‐laden mechanically heterogeneous microporous scaffolds is also demonstrated. This approach is particularly suited for the formation of modular, multimaterial scaffolds in situ, which could be applied to 3D bioprinting or to form more complex scaffolds to enhance regeneration of irregular wounds.     

Fermi Level Engineering of Passivation and Electron Transport Materials for p‐Type CuBi2O4 Employing a High‐Throughput Methodology

Metal oxide semiconductors are promising for solar photochemistry if the issues of excessive charge carrier recombination and material degradation can be resolved, which are both influenced by surface quality and interface chemistry. Coating the semiconductor with an overlayer to passivate surface states is a common remedial strategy but is less desirable than application of a functional coating that can improve carrier extraction and reduce recombination while mitigating corrosion. In this work, a data‐driven materials science approach utilizing high‐throughput methodologies, including inkjet printing and scanning droplet electrochemical cell measurements, is used to create and evaluate multi‐element coating libraries to discover new classes of candidate passivation and electron‐selective contact materials for p‐type CuBi₂O₄. The optimized overlayer (Cu_1.5TiO_z) improves the onset potential by 110 mV, the photocurrent by 2.8×, and the absorbed photon‐to‐current efficiency by 15.5% compared to non‐coated photoelectrodes. It is shown that these enhancements are related to reduced surface recombination through passivation of surface defect states as well as improved carrier extraction efficiency through Fermi level engineering. This work presents a generalizable, high‐throughput method to design and optimize passivation materials for a variety of semiconductors, providing a powerful platform for development of high‐performance photoelectrodes for incorporation into solar‐fuel generation systems.