De Novo Design of Integrin α5ß1 Modulating Proteins for Regenerative Medicine.

Integrin α5ß1 is crucial for cell attachment and migration in development and tissue regeneration, and α5ß1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5ß1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5ß1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.

One-Step Purification and N-Terminal Functionalization of Bioactive Proteins via Atypically Split Inteins.

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (ß-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.

Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks.

Biomechanical contributions of the ECM underpin cell growth and proliferation, differentiation, signal transduction, and other fate decisions. As such, biomaterials whose mechanics can be spatiotemporally altered - particularly in a reversible manner - are extremely valuable for studying these mechanobiological phenomena. Herein, we introduce a poly(ethylene glycol) (PEG)-based hydrogel model consisting of two interpenetrating step-growth networks that are independently formed via largely orthogonal bioorthogonal chemistries and sequentially degraded with distinct bacterial transpeptidases, affording reversibly tunable stiffness ranges that span healthy and diseased soft tissues (e.g., 500 Pa - 6 kPa) alongside terminal cell recovery for pooled and/or single-cell analysis in a near "biologically invisible" manner. Spatiotemporal control of gelation within the primary supporting network was achieved via mask-based and two-photon lithography; these stiffened patterned regions could be subsequently returned to the original soft state following sortase-based secondary network degradation. Using this approach, we investigated the effects of 4D-triggered network mechanical changes on human mesenchymal stem cell (hMSC) morphology and Hippo signaling, as well as Caco-2 colorectal cancer cell mechanomemory at the global transcriptome level via RNAseq. We expect this platform to be of broad utility for studying and directing mechanobiological phenomena, patterned cell fate, as well as disease resolution in softer matrices.

Highly Parallel Tissue Grafting for Combinatorial In Vivo Screening.

Material- and cell-based technologies such as engineered tissues hold great promise as human therapies. Yet, the development of many of these technologies becomes stalled at the stage of pre-clinical animal studies due to the tedious and low-throughput nature of in vivo implantation experiments. We introduce a 'plug and play' in vivo screening array platform called Highly Parallel Tissue Grafting (HPTG). HPTG enables parallelized in vivo screening of 43 three-dimensional microtissues within a single 3D printed device. Using HPTG, we screen microtissue formations with varying cellular and material components and identify formulations that support vascular self-assembly, integration and tissue function. Our studies highlight the importance of combinatorial studies that vary cellular and material formulation variables concomitantly, by revealing that inclusion of stromal cells can "rescue" vascular self-assembly in manner that is material-dependent. HPTG provides a route for accelerating pre-clinical progress for diverse medical applications including tissue therapy, cancer biomedicine, and regenerative medicine.

User-Controlled 4D Biomaterial Degradation with Substrate-Selective Sortase Transpeptidases for Single-Cell Biology.

Stimuli-responsive biomaterials show great promise for modeling disease dynamics ex vivo with spatiotemporal control over the cellular microenvironment. However, harvesting cells from such materials for downstream analysis without perturbing their state remains an outstanding challenge in 3/4-dimensional (3D/4D) culture and tissue engineering. In this manuscript, a fully enzymatic strategy for hydrogel degradation that affords spatiotemporal control over cell release while maintaining cytocompatibility is introduced. Exploiting engineered variants of the sortase transpeptidase evolved to recognize and selectively cleave distinct peptide sequences largely absent from the mammalian proteome, many limitations implicit to state-of-the-art methods to liberate cells from gels are sidestepped. It is demonstrated that evolved sortase exposure has minimal impact on the global transcriptome of primary mammalian cells and that proteolytic cleavage proceeds with high specificity; incorporation of substrate sequences within hydrogel crosslinkers permits rapid and selective cell recovery with high viability. In composite multimaterial hydrogels, it is shown that sequential degradation of hydrogel layers enables highly specific retrieval of single-cell suspensions for phenotypic analysis. It is expected that the high bioorthogonality and substrate selectivity of the evolved sortases will lead to their broad adoption as an enzymatic material dissociation cue and that their multiplexed use will enable newfound studies in 4D cell culture.

Substrate-Independent Micropatterning of Polymer Brushes Based on Photolytic Deactivation of Chemical Vapor Deposition Based Surface-Initiated Atom-Transfer Radical Polymerization Initiator Films.

Precise microscale arrangement of biomolecules and cells is essential for tissue engineering, microarray development, diagnostic sensors, and fundamental research in the biosciences. Biofunctional polymer brushes have attracted broad interest in these applications. However, patterning approaches to creating microstructured biointerfaces based on polymer brushes often involve tedious, expensive, and complicated procedures that are specifically designed for model substrates. We report a substrate-independent, facile, and scalable technique with which to prepare micropatterned biofunctional brushes with the ability to generate binary chemical patterns. Employing chemical vapor deposition (CVD) polymerization, a functionalized polymer coating decorated with 2-bromoisobutyryl groups that act as atom-transfer radical polymerization (ATRP) initiators was prepared and subsequently modified using UV light. The exposure of 2-bromoisobutyryl groups to UV light with wavelengths between 187 and 254 nm resulted in selective debromination, effectively eliminating the initiation of ATRP. In addition, when coatings incorporating both 2-bromoisobutyryl and primary amine groups were irradiated with UV light, the amines retained their functionality after UV treatment and could be conjugated to activated esters, facilitating binary chemical patterns. In contrast, polymer brushes were selectively grown from areas protected from UV treatment, as confirmed by atomic force microscopy, time-of-flight secondary ion mass spectrometry, and imaging ellipsometry. Furthermore, spatial control over biomolecular adhesion was achieved in three ways: (1) patterned nonfouling brushes resulted in nonspecific protein adsorption to areas not covered with polymer brushes; (2) patterned brushes decorated with active binding sides gave rise to specific protein immobilization on areas presenting polymer brushes; (3) and primary amines were co-patterned along with clickable polymer brushes bearing pendant alkyne groups, leading to bifunctional reactivity. Because this novel technique is independent of the original substrate's physicochemical properties, it can be extended to technologically relevant substrates such as polystyrene, polydimethylsiloxane, polyvinyl chloride, and steel. With further work, the photolytic deactivation of CVD-based initiator coatings promises to advance the utility of patterned biofunctional polymer brushes across a spectrum of biomedical applications.

Examining Nanoparticle Adsorption on Electrostatically "Patchy" Glycopolymer Brushes Using Real-Time ζ-Potential Measurements.

Biomaterial surfaces can possess chemical, topographical, or electrostatic heterogeneity, which can profoundly influence their performance. By developing experimental models that reliably simulate this nanoscale heterogeneity, we can predict how heterogeneous surfaces are transformed by their interactions with the dynamic physiological environment. In this work, we present a model surface where well-defined glycopolymer brushes are interspersed with positively charged binding sites, giving rise to an interface presenting a mixture of repulsive and adhesive cues to an approaching virus particle. We show that the density of the affinity sites relative to the glycopolymer brushes can be tuned precisely by modifying the chemical vapor deposition (CVD) copolymerization conditions. Further, we examined the effects of binding site density and glycopolymer brush architecture on the adsorption kinetics of virus-like nanoparticles through a novel approach employing time-resolved ζ-potential measurements. Most materials have charge-bearing, dynamic surfaces that are sensitive to electrostatic effects. Hence, adsorption-triggered changes in ζ-potential measurements can be captured in real time to monitor interfacial events. Real-time ζ-potential measurements present an interesting platform to probe the structure and function of chemically and electrostatically heterogeneous polymer interfaces. To validate this electrokinetic method, we examined the effect of neutravidin concentration on its rate of binding to biotinylated surfaces using ζ-potential and compared our results with QCM studies. By applying electrokinetic methods to examine the roles of glycopolymer brush architecture and surface charge of these tunable glycopolymer coatings, we can enhance our understanding of the interactions of viruses with heterogeneous biomaterial interfaces.