Effect of polar amino acid incorporation on Fmoc-diphenylalanine-based tetrapeptides.

Peptide hydrogels show great promise as extracellular matrix mimics due to their tuneable, fibrous nature. Through incorporation of polar cationic, polar anionic or polar neutral amino acids into the Fmoc-diphenylalanine motif, we show that electrostatic charge plays a key role in the properties of the subsequent gelators. Specifically, we show that an inverse relationship exists for biocompatibility in the solution state versus the gel state for cationic and anionic peptides. Finally, we use tethered bilayer lipid membrane (tBLM) experiments to suggest a likely mode of cytotoxicity for tetrapeptides which exhibit cytotoxicity in the solution state.

Kinetically Controlled Lifetimes in Redox-Responsive Transient Supramolecular Hydrogels.

It remains challenging to program soft materials to show dynamic, tunable time-dependent properties. In this work, we report a strategy to design transient supramolecular hydrogels based on kinetic control of competing reactions. Specifically, the pH-triggered self-assembly of a redox-active supramolecular gelator, N,N'-dibenzoyl-l-cystine (DBC) in the presence of a reducing agent, which acts to disassemble the system. The lifetimes of the transient hydrogels can be tuned simply by pH or reducing agent concentration. We find through kinetic analysis that gel formation hinders the ability of the reducing agent and enables longer transient hydrogel lifetimes than would be predicted. The transient hydrogels undergo clean cycles, with no kinetically trapped aggregates observed. As a result, multiple transient hydrogel cycles are demonstrated and can be predicted. This work contributes to our understanding of designing transient assemblies with tunable temporal control.

Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

A Capped Dipeptide Which Simultaneously Exhibits Gelation and Crystallization Behavior.

Short peptides capped at their N-terminus are often highly efficient gelators, yet notoriously difficult to crystallize. This is due to strong unidirectional interactions within fibers, resulting in structure propagation only along one direction. Here, we synthesize the N-capped dipeptide, benzimidazole-diphenylalanine, which forms both hydrogels and single crystals. Even more remarkably, we show using atomic force microscopy the coexistence of these two distinct phases. We then use powder X-ray diffraction to investigate whether the single crystal structure can be extrapolated to the molecular arrangement within the hydrogel. The results suggest parallel ß-sheet arrangement as the dominant structural motif, challenging existing models for gelation of short peptides, and providing new directions for the future rational design of short peptide gelators.

Effect of heterocyclic capping groups on the self-assembly of a dipeptide hydrogel.

The mechanism and design rules associated with the self-assembly of short peptides into hydrogels is currently not well understood. In this work, four diphenylalanine-based peptides have been synthesised, bearing heterocyclic capping groups which have different degrees of hydrogen bonding potential and nitrogen substitution. For these four peptides, zeta potential and electrical impedance spectroscopy measurements were undertaken to monitor gelation, with the impedance data showing different gelation times for each peptide hydrogel. Through a combination of atomic force microscopy and rheological measurmeents, including dynamic strain and frequency sweeps, and thixotropic tests, the relationship between the mechanism of self-assembly in these hydrogels and their macroscopic behaviour can be established. It is observed that the degree of nitrogen substitution affects the self-assembly mechanisms of the hydrogels and as such, that there is an interplay between branching and bundling self-assembly pathways that are responsible for the final properties of each hydrogel.

Quantifying highly efficient incoherent energy transfer in perylene-based multichromophore arrays.

Multichromophore perylene arrays were designed and synthesized to have extremely efficient resonance energy transfer. Using broadband ultrafast photoluminescence and transient absorption spectroscopies, transfer timescales of approximately 1 picosecond were resolved, corresponding to efficiencies of up to 99.98%. The broadband measurements also revealed spectra corresponding to incoherent transfer between localized states. Polarization resolved spectroscopy was used to measure the dipolar angles between donor and acceptor chromophores, thereby enabling geometric factors to be fixed when assessing the validity of Förster theory in this regime. Förster theory was found to predict the correct magnitude of transfer rates, with measured ∼2-fold deviations consistent with the breakdown of the point-dipole approximation at close approach. The materials presented, along with the novel methods for quantifying ultrahigh energy transfer efficiencies, will be valuable for applications demanding extremely efficient energy transfer, including fluorescent solar concentrators, optical gain, and photonic logic devices.

4D Multimaterial Printing of Soft Actuators with Spatial and Temporal Control.

Soft actuators (SAs) are devices which can interact with delicate objects in a manner not achievable with traditional robotics. While it is possible to design a SA whose actuation is triggered via an external stimulus, the use of a single stimulus creates challenges in the spatial and temporal control of the actuation. Herein, a 4D printed multimaterial soft actuator design (MMSA) whose actuation is only initiated by a combination of triggers (i.e., pH and temperature) is presented. Using 3D printing, a multilayered soft actuator with a hydrophilic pH-sensitive layer, and a hydrophobic magnetic and temperature-responsive shape-memory polymer layer, is designed. The hydrogel responds to environmental pH conditions by swelling or shrinking, while the shape-memory polymer can resist the shape deformation of the hydrogel until triggered by temperature or light. The combination of these stimuli-responsive layers allows for a high level of spatiotemporal control of the actuation. The utility of the 4D MMSA is demonstrated via a series of cargo capture and release experiments, validating its ability to demonstrate active spatiotemporal control. The MMSA concept provides a promising research direction to develop multifunctional soft devices with potential applications in biomedical engineering and environmental engineering.

3,4-Ethylenedioxythiophene Hydrogels: Relating Structure and Charge Transport in Supramolecular Gels.

Ionic charge transport is a ubiquitous language of communication in biological systems. As such, bioengineering is in constant need of innovative, soft, and biocompatible materials that facilitate ionic conduction. Low molecular weight gelators (LMWGs) are complex self-assembled materials that have received increasing attention in recent years. Beyond their biocompatible, self-healing, and stimuli responsive facets, LMWGs can be viewed as a "solid" electrolyte solution. In this work, we investigate 3,4-ethylenedioxythiophene (EDOT) as a capping group for a small peptide library, which we use as a system to understand the relationship between modes of assembly and charge transport in supramolecular gels. Through a combination of techniques including small-angle neutron scattering (SANS), NMR-based Van't Hoff analysis, atomic force microscopy (AFM), rheology, four-point probe, and electrochemical impedance spectroscopy (EIS), we found that modifications to the peptide sequence result in distinct assembly pathways, thermodynamic parameters, mechanical properties, and ionic conductivities. Four-point probe conductivity measurements and electrochemical impedance spectroscopy suggest that ionic conductivity is approximately doubled by programmable gel assemblies with hollow cylinder morphologies relative to gels containing solid fibers or a control electrolyte. More broadly, it is hoped this work will serve as a platform for those working on charge transport of aqueous soft materials in general.

Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis.

The intrinsic heterogeneity of many nanoformulations is currently challenging to characterize on both the single particle and population level. Therefore, there is great opportunity to develop advanced techniques to describe and understand nanomedicine heterogeneity, which will aid translation to the clinic by informing manufacturing quality control, characterization for regulatory bodies, and connecting nanoformulation properties to clinical outcomes to enable rational design. Here, we present an analytical technique to provide such information, while measuring the nanocarrier and cargo simultaneously with label-free, nondestructive single particle automated Raman trapping analysis (SPARTA). We first synthesized a library of model compounds covering a range of hydrophilicities and providing distinct Raman signals. These compounds were then loaded into model nanovesicles (polymersomes) that can load both hydrophobic and hydrophilic cargo into the membrane or core regions, respectively. Using our analytical framework, we characterized the heterogeneity of the population by correlating the signal per particle from the membrane and cargo. We found that core and membrane loading can be distinguished, and we detected subpopulations of highly loaded particles in certain cases. We then confirmed the suitability of our technique in liposomes, another nanovesicle class, including the commercial formulation Doxil. Our label-free analytical technique precisely determines cargo location alongside loading and release heterogeneity in nanomedicines, which could be instrumental for future quality control, regulatory body protocols, and development of structure-function relationships to bring more nanomedicines to the clinic.

Use of pyridazinediones for tuneable and reversible covalent cysteine modification applied to peptides, proteins and hydrogels.

Reversible cysteine modification has been found to be a useful tool for a plethora of applications such as selective enzymatic inhibition, activity-based protein profiling and/or cargo release from a protein or a material. However, only a limited number of reagents display reliable dynamic/reversible thiol modification and, in most cases, many of these reagents suffer from issues of stability, a lack of modularity and/or poor rate tunability. In this work, we demonstrate the potential of pyridazinediones as novel reversible and tuneable covalent cysteine modifiers. We show that the electrophilicity of pyridazinediones correlates to the rates of the Michael addition and retro-Michael deconjugation reactions, demonstrating that pyridazinediones provide an enticing platform for readily tuneable and reversible thiol addition/release. We explore the regioselectivity of the novel reaction and unveil the reason for the fundamental increased reactivity of aryl bearing pyridazinediones by using DFT calculations and corroborating findings with SCXRD. We also applied this fundamental discovery to making more rapid disulfide rebridging agents in related work. We finally provide the groundwork for potential applications in various areas with exemplification using readily functionalised "clickable" pyridazinediones on clinically relevant cysteine and disulfide conjugated proteins, as well as on a hydrogel material.

Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

Tunable Microgel-Templated Porogel (MTP) Bioink for 3D Bioprinting Applications.

Micropores are essential for tissue engineering to ensure adequate mass transportation for embedded cells. Despite the considerable progress made by advanced 3D bioprinting technologies, it remains challenging to engineer micropores of 100 µm or smaller in cell-laden constructs. Here, a microgel-templated porogel (MTP) bioink platform is reported to introduce controlled microporosity in 3D bioprinted hydrogels in the presence of living cells. Templated gelatin microgels are fabricated with varied sizes (≈10, ≈45, and ≈100 µm) and mixed with photo-crosslinkable formulations to make composite MTP bioinks. The addition of microgels significantly enhances the shear-thinning and self-healing viscoelastic properties and thus the printability of bioinks with cell densities up to 1 × 108 mL-1 in matrix. Consistent printability is achieved for a series of MTP bioinks based on different component ratios and matrix materials. After photo-crosslinking the matrix phase, the templated microgels dissociated and diffused under physiological conditions, resulting in corresponding micropores in situ. When embedding osteoblast-like cells in the matrix phase, the MTP bioinks support higher metabolic activity and more uniform mineral formation than bulk gel controls. The approach provides a facile strategy to engineer precise micropores in 3D printed structures to compensate for the limited resolution of current bioprinting approaches.

Design and clinical application of injectable hydrogels for musculoskeletal therapy.

Musculoskeletal defects are an enormous healthcare burden and source of pain and disability for individuals. With an aging population, the proportion of individuals living with these medical indications will increase. Simultaneously, there is pressure on healthcare providers to source efficient solutions, which are cheaper and less invasive than conventional technology. This has led to an increased research focus on hydrogels as highly biocompatible biomaterials that can be delivered through minimally invasive procedures. This review will discuss how hydrogels can be designed for clinical translation, particularly in the context of the new European Medical Device Regulation (MDR). We will then do a deep dive into the clinically used hydrogel solutions that have been commercially approved or have undergone clinical trials in Europe or the United States. We will discuss the therapeutic mechanism and limitations of these products. Due to the vast application areas of hydrogels, this work focuses only on treatments of cartilage, bone, and the nucleus pulposus. Lastly, the main steps toward clinical translation of hydrogels as medical devices are outlined. We suggest a framework for how academics can assist small and medium MedTech enterprises conducting the initial clinical investigation and post-market clinical follow-up required in the MDR. It is evident that the successful translation of hydrogels is governed by acquiring high-quality pre-clinical and clinical data confirming the device mechanism of action and safety.

A dynamic duo.

Self-assembly of nanofibers facilitates the repair of spinal cord injury in mice.

High-Throughput Peptide Derivatization toward Supramolecular Diversification in Microtiter Plates.

The evolution of life on earth eventually leads to the emergence of species with increased complexity and diversity. Similarly, evolutionary chemical space exploration in the laboratory is a key step to pursue the structural and functional diversity of supramolecular systems. Here, we present a powerful tool that enables rapid peptide diversification and employ it to expand the chemical space for supramolecular functions. Central to this strategy is the exploitation of palladium-catalyzed Suzuki-Miyaura cross-coupling reactions to direct combinatorial synthesis of peptide arrays in microtiter plates under an open atmosphere. Taking advantage of this in situ library design, our results unambiguously deliver a fertile platform for creating a set of intriguing peptide functions including green fluorescent protein-like peptide emitters with chemically encoded emission colors, hierarchical self-assembly into nano-objects, and macroscopic hydrogels. This work also offers opportunities for quickly surveying the diversified peptide arrays and thereby identifying the structural factors that modulate peptide properties.

Correction: Non-reversible heat-induced gelation of a biocompatible Fmoc-hexapeptide in water.

Correction for 'Non-reversible heat-induced gelation of a biocompatible Fmoc-hexapeptide in water' by Jonathan P. Wojciechowski et al., Nanoscale, 2020, 12, 8262-8267, DOI: .

Non-reversible heat-induced gelation of a biocompatible Fmoc-hexapeptide in water.

Hydrogel materials which respond to changes in temperature are widely applicable for injectable drug delivery or tissue engineering applications. Here, we report the unsual heat-induced gelation behaviour of a low molecular weight gelator based on an Fmoc-hexapeptide, Fmoc-GFFRGD. We show that Fmoc-GFFRGD forms kinetically stable fibres when mixed with divalent cations (e.g. Ca2+). Gelation of the mixture occurs upon heating of the mixture which enables electrostatic screening by the divalent cations and hydrophobic collapse of the fibres to give a self-supporting hydrogel network that shows good biocompatibility with L929 fibroblast cells. This work highlights a unique mechanism to initiate heat-induced gelation which should find opportunities as a gelation trigger for injectable hydrogels or fundamental self-assembly applications.

Ultrasound-Triggered Enzymatic Gelation.

Hydrogels are formed using various triggers, including light irradiation, pH adjustment, heating, cooling, or chemical addition. Here, a new method for forming hydrogels is introduced: ultrasound-triggered enzymatic gelation. Specifically, ultrasound is used as a stimulus to liberate liposomal calcium ions, which then trigger the enzymatic activity of transglutaminase. The activated enzyme catalyzes the formation of fibrinogen hydrogels through covalent intermolecular crosslinking. The catalysis and gelation processes are monitored in real time and both the enzyme kinetics and final hydrogel properties are controlled by varying the initial ultrasound exposure time. This technology is extended to microbubble-liposome conjugates, which exhibit a stronger response to the applied acoustic field and are also used for ultrasound-triggered enzymatic hydrogelation. To the best of the knowledge, these results are the first instance in which ultrasound is used as a trigger for either enzyme catalysis or enzymatic hydrogelation. This approach is highly versatile and can be readily applied to different ion-dependent enzymes or gelation systems. Moreover, this work paves the way for the use of ultrasound as a remote trigger for in vivo hydrogelation.

Expanding and optimizing 3D bioprinting capabilities using complementary network bioinks.

A major challenge in three-dimensional (3D) bioprinting is the limited number of bioinks that fulfill the physicochemical requirements of printing while also providing a desirable environment for encapsulated cells. Here, we address this limitation by temporarily stabilizing bioinks with a complementary thermo-reversible gelatin network. This strategy enables the effective printing of biomaterials that would typically not meet printing requirements, with instrument parameters and structural output largely independent of the base biomaterial. This approach is demonstrated across a library of photocrosslinkable bioinks derived from natural and synthetic polymers, including gelatin, hyaluronic acid, chondroitin sulfate, dextran, alginate, chitosan, heparin, and poly(ethylene glycol). A range of complex and heterogeneous structures are printed, including soft hydrogel constructs supporting the 3D culture of astrocytes. This highly generalizable methodology expands the palette of available bioinks, allowing the biofabrication of constructs optimized to meet the biological requirements of cell culture and tissue engineering.

Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation.

The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.