Low-cost, versatile, and highly reproducible microfabrication pipeline to generate 3D-printed customised cell culture devices with complex designs.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the µm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.

Expanding the scope of the successive ring expansion strategy for macrocycle and medium-sized ring synthesis: unreactive and reactive lactams.

New methods are described that expand the scope of the Successive Ring Expansion (SuRE) with respect to synthetically challenging lactams. A protocol has been developed for use with 'unreactive' lactams, enabling SuRE reactions to be performed on subsrates that fail under previously established conditions. Ring expansion is also demonstarted on 'reactive' lactams derived from iminosugars for the first time. The new SuRE methods were used to prepare a diverse array of medium-sized and macrocyclic lactams and lactones, which were evaluted in an anti-bacterial assay against E. coli BW25113WT.

Strain-Promoted Cycloadditions in Lipid Bilayers Triggered by Liposome Fusion.

Due to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide-alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide- and strained alkyne-functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays.

Crossing the Solubility Rubicon: 15-Crown-5 Facilitates the Preparation of Water-Soluble Sulfo-NHS Esters in Organic Solvents.

The Sulfo-NHS ester is a mainstay reagent for facilitating amide bond formation between carboxylic acids and amine functionalities in water. However, the preparation of Sulfo-NHS esters currently requires hydrophobic carboxylic acids, which are poorly water-soluble, to first be reacted with the N-hydroxysulfosuccinimide sodium salt, which is insoluble in organic solvents. The mutually incompatible solvation requirements thus complicate the synthesis of Sulfo-NHS esters. As a simple, rapid, and cost-effective solution to this problem, we report that the use of 15-crown-5 to complex the sodium cation of N-hydroxysulfosuccinimide sodium salt circumnavigates these solvation incompatibility issues by rendering the N-hydroxysulfosuccinimide salt soluble in organic solvents, resulting in a cleaner esterification reaction and thus improved yields of activated ester product. We also demonstrate that the resultant "crowned" Sulfo-NHS-ester remains water-soluble and is no less reactive than its classic "uncrowned" Sulfo-NHS counterpart when used in bioconjugation reactions between protein amine-functionalities and hydrophobic carboxylic acids.

Hydrogel Cross-Linking via Thiol-Reactive Pyridazinediones.

Thiol-reactive Michael acceptors are commonly used for the formation of chemically cross-linked hydrogels. In this paper, we address the drawbacks of many Michael acceptors by introducing pyridazinediones as new cross-linking agents. Through the use of pyridazinediones and their mono- or dibrominated analogues, we show that the mechanical strength, swelling ratio, and rate of gelation can all be controlled in a pH-sensitive manner. Moreover, we demonstrate that the degradation of pyridazinedione-gels can be induced by the addition of thiols, thus providing a route to responsive or dynamic gels, and that monobromo-pyridazinedione gels are able to support the proliferation of human cells. We anticipate that our results will provide a valuable and complementary addition to the existing toolkit of cross-linking agents, allowing researchers to tune and rationally design the properties of biomedical hydrogels.

Selectivity and stability of N-terminal targeting protein modification chemistries.

Protein N-termini provide uniquely reactive motifs for single site protein modification. Though a number of reactions have been developed to target this site, the selectivity, generality, and stability of the conjugates formed has not been studied. We have therefore undertaken a comprehensive comparative study of the most promising methods for N-terminal protein modification, and find that there is no 'one size fits all' approach, necessitating reagent screening for a particular protein or application. Moreover, we observed limited stability in all cases, leading to a need for continued innovation and development in the bioconjugation field.

Preparation and Application of an Inexpensive α-Formylglycine Building Block Compatible with Fmoc Solid-Phase Peptide Synthesis.

α-Formylglycine (fGly) is a rare residue located in the active site of sulfatases and serves as a precursor to pharmaceutically relevant motifs. The installation of fGly motifs into peptides is currently challenging due to degradation under the acidic and nucleophile-rich conditions accompanying resin cleavage during solid-phase peptide synthesis. We report the synthesis of acid- and nucleophile-tolerant α-formylglycine building blocks from vitamin C and use them to prepare callyaerin A, a macrocyclic peptide containing an fGly-derived motif.

Insight into ortho-boronoaldehyde conjugation via a FRET-based reporter assay.

Ortho-boronoaldehydes react with amine-based nucleophiles with dramatically increased rates and product stabilities, relative to unfunctionalised benzaldehydes, leading to exciting applications across biological and material chemistry. We have developed a novel Förster resonance energy transfer (FRET)-based assay to provide key new insights into the reactivity of these boronoaldehydes, allowing us to track conjugation with unprecedented sensitivity and accuracy under standardised conditions. Our results highlight the key role played by reaction pH, buffer additives, and boronoaldehyde structure in controlling conjugation speed and stability, providing design criteria for further innovations and applications in the field.

Nuclear Magnetic Resonance and Metadynamics Simulations Reveal the Atomistic Binding of l-Serine and O-Phospho-l-Serine at Disordered Calcium Phosphate Surfaces of Biocements.

Interactions between biomolecules and structurally disordered calcium phosphate (CaP) surfaces are crucial for the regulation of bone mineralization by noncollagenous proteins, the organization of complexes of casein and amorphous calcium phosphate (ACP) in milk, as well as for structure-function relationships of hybrid organic/inorganic interfaces in biomaterials. By a combination of advanced solid-state NMR experiments and metadynamics simulations, we examine the detailed binding of O-phospho-l-serine (Pser) and l-serine (Ser) with ACP in bone-adhesive CaP cements, whose capacity of gluing fractured bone together stems from the close integration of the organic molecules with ACP over a subnanometer scale. The proximity of each carboxy, aliphatic, and amino group of Pser/Ser to the Ca2+ and phosphate species of ACP observed from the metadynamics-derived models agreed well with results from heteronuclear solid-state NMR experiments that are sensitive to the 13C-31P and 15N-31P distances. The inorganic/organic contacts in Pser-doped cements are also contrasted with experimental and modeled data on the Pser binding at nanocrystalline HA particles grown from a Pser-bearing aqueous solution. The molecular adsorption is driven mainly by electrostatic interactions between the negatively charged carboxy/phosphate groups and Ca2+ cations of ACP, along with H bonds to either protonated or nonprotonated inorganic phosphate groups. The Pser and Ser molecules anchor at their phosphate/amino and carboxy/amino moieties, respectively, leading to an extended molecular conformation across the surface, as opposed to an "upright standing" molecule that would result from the binding of one sole functional group.

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.

Photochemical Methods for Peptide Macrocyclisation.

Photochemical reactions have been the subject of renewed interest over the last two decades, leading to the development of many new, diverse and powerful chemical transformations. More recently, these developments have been expanded to enable the photochemical macrocyclisation of peptides and small proteins. These constructs benefit from increased stability, structural rigidity and biological potency over their linear counterparts, providing opportunities for improved therapeutic agents. In this review, an overview of both the established and emerging methods for photochemical peptide macrocyclisation is presented, highlighting both the limitations and opportunities for further innovation in the field.

Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery.

Many diseases are associated with the dysregulated activity of enzymes, such as matrix metalloproteinases (MMPs). This dysregulation can be leveraged in drug delivery to achieve disease- or site-specific cargo release. Self-assembled polymeric nanoparticles are versatile drug carrier materials due to the accessible diversity of polymer chemistry. However, efficient loading of sensitive cargo, such as proteins, and introducing functional enzyme-responsive behaviour remain challenging. Herein, peptide-crosslinked, temperature-sensitive nanogels for protein delivery were designed to respond to MMP-7, which is overexpressed in many pathologies including cancer and inflammatory diseases. The incorporation of N-cyclopropylacrylamide (NCPAM) into N-isopropylacrylamide (NIPAM)-based copolymers enabled us to tune the polymer lower critical solution temperature from 33 to 44 °C, allowing the encapsulation of protein cargo and nanogel-crosslinking at slightly elevated temperatures. This approach resulted in nanogels that were held together by MMP-sensitive peptides for enzyme-specific protein delivery. We employed a combination of cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small angle neutron scattering (SANS), and fluorescence correlation spectroscopy (FCS) to precisely decipher the morphology, self-assembly mechanism, enzyme-responsiveness, and model protein loading/release properties of our nanogel platform. Simple variation of the peptide linker sequence and combining multiple different crosslinkers will enable us to adjust our platform to target specific diseases in the future.

Synthesis of Phospho-Amino Acid Analogues as Tissue Adhesive Cement Additives.

In this paper we report the synthesis of a library of phospho-amino acid analogues, via a novel single-step allyl-phosphoester protection/Pd-mediated deprotection strategy. These phosphoserine and phosphotyrosine analogues were then applied as additives to create adhesive calcium phosphate cements, allowing us to probe the chemical origins of the increased surface binding strength. We demonstrate the importance of multiple calcium binding motifs in mediating adhesion, as well as highlighting the crucial role played by substrate hydrophobicity and orientation in controlling binding strength.

An Electroactive Oligo-EDOT Platform for Neural Tissue Engineering.

The unique electrochemical properties of the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) make it an attractive material for use in neural tissue engineering applications. However, inadequate mechanical properties, and difficulties in processing and lack of biodegradability have hindered progress in this field. Here, the functionality of PEDOT:PSS for neural tissue engineering is improved by incorporating 3,4-ethylenedioxythiophene (EDOT) oligomers, synthesized using a novel end-capping strategy, into block co-polymers. By exploiting end-functionalized oligoEDOT constructs as macroinitiators for the polymerization of poly(caprolactone), a block co-polymer is produced that is electroactive, processable, and bio-compatible. By combining these properties, electroactive fibrous mats are produced for neuronal culture via solution electrospinning and melt electrospinning writing. Importantly, it is also shown that neurite length and branching of neural stem cells can be enhanced on the materials under electrical stimulation, demonstrating the promise of these scaffolds for neural tissue engineering.

Shape-controlled synthesis and in situ characterisation of anisotropic Au nanomaterials using liquid cell transmission electron microscopy.

Understanding the mechanisms behind crystal nucleation and growth is a fundamental requirement for the design and production of bespoke nanomaterials with controlled sizes and morphologies. Herein, we select gold (Au) nanoparticles as the model system for our study due to their representative applications in biology, electronics and optoelectronics. We investigate the radiation-induced in situ growth of gold (Au) particles using liquid cell transmission electron microscopy (LCTEM) and study the growth kinetics of non-spherical Au structures. Under controlled electron fluence, liquid flow rate and Au3+ ion supply, we show the favoured diffusion-limited growth of multi-twinned nascent Au seed particles into branched structures when using thin liquid cells (100 nm and 250 nm) in LCTEM, whereas faceted structures (e.g., spheres, rods, and prisms) formed when using a 1 µm thick liquid cell. In addition, we observed that anisotropic Au growth could be modulated by Au-binding amyloid fibrils, which we ascribe to their capability to regulate Au3+ ion diffusion and mass transfer in solution. We anticipate that this study will provide new perspectives on the shape-controlled synthesis of anisotropic metallic nanomaterials using LCTEM.

A Novel Class of Injectable Bioceramics that Glue Tissues and Biomaterials.

Calcium phosphate cements (CPCs) are clinically effective void fillers that are capable of bridging calcified tissue defects and facilitating regeneration. However, CPCs are completely synthetic/inorganic, unlike the calcium phosphate that is found in calcified tissues, and they lack an architectural organization, controlled assembly mechanisms, and have moderate biomechanical strength, which limits their clinical effectiveness. Herein, we describe a new class of bioinspired CPCs that can glue tissues together and bond tissues to metallic and polymeric biomaterials. Surprisingly, alpha tricalcium phosphate cements that are modified with simple phosphorylated amino acid monomers of phosphoserine (PM-CPCs) bond tissues up to 40-fold stronger (2.5⁻4 MPa) than commercial cyanoacrylates (0.1 MPa), and 100-fold stronger than surgical fibrin glue (0.04 MPa), when cured in wet-field conditions. In addition to adhesion, phosphoserine creates other novel properties in bioceramics, including a nanoscale organic/inorganic composite microstructure, and templating of nanoscale amorphous calcium phosphate nucleation. PM-CPCs are made of the biocompatible precursors calcium, phosphate, and amino acid, and these represent the first amorphous nano-ceramic composites that are stable in liquids.

Palladium-mediated enzyme activation suggests multiphase initiation of glycogenesis.

Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi1, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease2-4, and mouse knockout5 and inherited human mutations6 of GYG impair glycogen synthesis. GYG acts as a 'seed core' for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195. Precise mechanistic studies have so far been prevented by an inability to access homogeneous glycoforms of this protein, which unusually acts as both catalyst and substrate. Here we show that unprecedented direct access to different, homogeneously glucosylated states of GYG can be accomplished through a palladium-mediated enzyme activation 'shunt' process using on-protein C-C bond formation. Careful mimicry of GYG intermediates recapitulates catalytic activity at distinct stages, which in turn allows discovery of triphasic kinetics and substrate plasticity in GYG's use of sugar substrates. This reveals a tolerant but 'proof-read' mechanism that underlies the precision of this metabolic process. The present demonstration of direct, chemically controlled access to intermediate states of active enzymes suggests that such ligation-dependent activation could be a powerful tool in the study of mechanism.

Duplex-Specific Nuclease-Amplified Detection of MicroRNA Using Compact Quantum Dot-DNA Conjugates.

Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances in functional nanoparticles, DNA nanotechnology, and nuclease-enzyme-based amplification can give rise to new assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple DNA probes, and the enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where a single miRNA target triggers the cleavage of many DNA signal probes. The incorporation of DNA-functionalized QDs enabled a quantitative fluorescent readout, mediated by Förster resonance energy transfer (FRET)-based interaction with the DNA signal probes. Our approach splits the reaction in two, performing the enzyme-mediated amplification and QD-based detection steps separately such that each reaction could be optimized for performance of the active components. Target recycling gave ca. 3 orders of magnitude amplification, yielding highly sensitive detection with a limit of 42 fM (or 1.2 amol) of miR-148, with excellent selectivity versus mismatched sequences and other miRNAs. Furthermore, we used an alternative target (miR-21) and FRET pair for direct and absolute quantification of miR-21 in RNA extracts from human cancer and normal cell lines.

Achieving Controlled Biomolecule-Biomaterial Conjugation.

The conjugation of biomolecules can impart materials with the bioactivity necessary to modulate specific cell behaviors. While the biological roles of particular polypeptide, oligonucleotide, and glycan structures have been extensively reviewed, along with the influence of attachment on material structure and function, the key role played by the conjugation strategy in determining activity is often overlooked. In this review, we focus on the chemistry of biomolecule conjugation and provide a comprehensive overview of the key strategies for achieving controlled biomaterial functionalization. No universal method exists to provide optimal attachment, and here we will discuss both the relative advantages and disadvantages of each technique. In doing so, we highlight the importance of carefully considering the impact and suitability of a particular technique during biomaterial design.