Hyperspectral Mapping of Human Primary and Stem Cells at Cell-Matrix Interfaces.

Extracellular matrices interface with cells to promote cell growth and tissue development. Given this critical role, matrix mimetics are introduced to enable biomedical materials ranging from tissue engineering scaffolds and tumor models to organoids for drug screening and implant surface coatings. Traditional microscopy methods are used to evaluate such materials in their ability to support exploitable cell responses, which are expressed in changes in cell proliferation rates and morphology. However, the physical imaging methods do not capture the chemistry of cells at cell-matrix interfaces. Herein, we report hyperspectral imaging to map the chemistry of human primary and embryonic stem cells grown on matrix materials, both native and artificial. We provide the statistical analysis of changes in lipid and protein content of the cells obtained from infrared spectral maps to conclude matrix morphologies as a major determinant of biochemical cell responses. The study demonstrates an effective methodology for evaluating bespoke matrix materials directly at cell-matrix interfaces.

Folding-Mediated DNA Delivery by α-Helical Amphipathic Peptides.

The renaissance gene therapy experiences these days requires specialist biomaterials and a systemic understanding of major factors influencing their ability to deliver genetic material. Peptide transfection systems represent a major class of such biomaterials. Several peptidic reagents have been commercialized to date. However, a comparative assessment of peptide sequences alone without auxiliary support or excipients against a common determinant for their ability to complex and deliver DNA has been lacking. This study cross-compares commercial and experimental transfection reagents from the same family of helical amphiphiles. Factors defining the efficacy of DNA delivery including cell uptake and gene expression are assessed along with cytotoxicity and DNA complexation. The results show that despite differences in sequence composition, length, and origin, peptide reagents of the same structural family exhibit similar characteristics and limitations with common variability trends. The cross-comparison revealed that functional DNA delivery is independent of the peptide sequence used but is mediated by the ability of the reagents to co-fold with DNA. Peptide folding proved to be the common determinant for DNA complexation and delivery by peptidic transfection reagents.

An SI-traceable reference material for virus-like particles.

A reference material for virus-like particles traceable to the International System of Units (Système International d'Unités - the SI) is reported. The material addresses the need for developing reference standards to benchmark virus-like gene delivery systems and help harmonize measurement approaches for characterization and testing. The material is a major component of synthetic polypeptide virus-like particles produced by the state-of-the-art synthetic and analytical chemistry methods used to generate gene delivery systems. The purity profile of the material is evaluated to the highest metrological order demonstrating traceability to the SI. The material adds to the emerging toolkit of reference standards for quantitative biology.

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics.

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions-their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide's physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

Investigating Membrane-Mediated Antimicrobial Peptide Interactions with Synchrotron Radiation Far-Infrared Spectroscopy.

Synchrotron radiation-based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio-molecular phenomena including folding-mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near-THz 400-40 cm-1 range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature-dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane-induced and folding-mediated activity of AMPs. The far-FTIR study offers a direct and information-rich probe of membrane-related antimicrobial interactions.

Atomic force microscopy to elucidate how peptides disrupt membranes.

Atomic force microscopy is an increasingly attractive tool to study how peptides disrupt membranes. Often performed on reconstituted lipid bilayers, it provides access to time and length scales that allow dynamic investigations with nanometre resolution. Over the last decade, AFM studies have enabled visualisation of membrane disruption mechanisms by antimicrobial or host defence peptides, including peptides that target malignant cells and biofilms. Moreover, the emergence of high-speed modalities of the technique broadens the scope of investigations to antimicrobial kinetics as well as the imaging of peptide action on live cells in real time. This review describes how methodological advances in AFM facilitate new insights into membrane disruption mechanisms.

Peptide Nanoparticles for Gene Packaging and Intracellular Delivery.

Efficient gene transfer is necessary for advanced biotechnologies ranging from gene therapy to synthetic biology. Peptide nanoparticles provide suitable packaging systems promoting targeted gene expression or silencing. Though these systems have yet to match the transfection efficacy of viruses, they are typically devoid of drawbacks characteristic of virus-based vectors, including insertional mutagenesis, low packaging capacities, and strong immune responses. Given the promise nanoparticle formulations hold for gene delivery, methods of their preparation and accurate analysis of their physicochemical and biological properties become indispensable for progress toward systems that seek to outperform viral vectors. Herein, we report a comprehensive protocol for the preparation and characterization of archetypal peptide nanoparticles resulting from nonspecific and noncovalent complexation with RNA and DNA.

Imaging and 3D Reconstruction of De Novo Peptide Capsids.

Nanoscale systems encapsulating biomacromolecules hold promise for cell and gene therapies. Common issues hampering progress include polydispersity, heterogeneity in size and shape, agglomeration, and poor stability. Much attention is given to the search of novel designs. However, reliable protocols for the validation of encapsulating systems in the continuum of their physicochemical properties, from design to ultrastructure, are lacking. Herein, we report electron microscopy protocols for biologically functional shell-like peptide capsids, which exhibit the physical characteristics of viruses including folding-mediated self-assembly, hollow shell morphology, and uniformity in size.

Imaging the Effects of Peptide Materials on Phospholipid Membranes by Atomic Force Microscopy.

Recent advances in biomolecular design require accurate measurements performed in native or near-native environments in real time. Atomic force microscopy (AFM) is a powerful tool to observe the dynamics of biologically relevant processes at aqueous interfaces with high spatial resolution. Here, we describe imaging protocols to characterize the effects of peptide materials on phospholipid membranes in solution by AFM. These protocols can be used to determine the mechanism and kinetics of membrane-associated activities at the nanoscale.

Ultramicrotomy Analysis of Peptide-Treated Cells.

Electron microscopy offers necessary precision for the characterization of peptide materials at the nanoscale. Analysis is typically performed for acellular material specimens, whereas measurements in more complex, cellular environments prompt additional considerations for sample processing. Herein, we describe a protocol for the ultramicrotomy analysis of peptide-treated bacterial and mammalian cells. An emphasis is made on cell analysis following peptide treatment, as opposed to peptide analysis in cells, and focuses on sample processing, including fixation and staining procedures, resin embedding, sectioning, and imaging. The application of the protocol is demonstrated for intracellular measurements using antimicrobial peptide materials.

Tracking Insulin Glycation in Real Time by Time-Resolved Emission Spectroscopy.

The application of time-resolved fluorescence sensing to the study of heterogenic biomolecular systems remains challenging because of the complexity of the resulting photophysics. Measuring the time-resolved emission spectroscopy (TRES) spectra can provide a more informative alternative to the modeling of the fluorescence decay that is currently employed. Here, we demonstrate this approach by monitoring real-time changes in intrinsic insulin fluorescence by TRES as a straightforward probe to directly measure kinetics of insulin aggregation and glycation. Our findings hold promise for monitoring the storage of insulin and its application in the control of diabetes and may support the development of more effective therapeutics against amyloidosis.

Helminth Defense Molecules as Design Templates for Membrane Active Antibiotics.

A design template for membrane active antibiotics against microbial and tumor cells is described. The template is an amino acid sequence that combines the properties of helminth defense molecules, which are not cytolytic, with the properties of host-defense peptides, which disrupt microbial membranes. Like helminth defense molecules, the template folds into an amphipathic helix in both mammalian host and microbial phospholipid membranes. Unlike these molecules, the template exhibits antimicrobial and anticancer properties that are comparable to those of antimicrobial and anticancer antibiotics. The selective antibiotic activity of the template builds upon a functional synergy between three distinctive faces of the helix, which is in contrast to two faces of membrane-disrupting amphipathic structures. This synergy enables the template to adapt pore formation mechanisms according to the nature of the target membrane, inducing the lysis of microbial and tumor cells.

Protein fibrillogenesis model tracked by its intrinsic time-resolved emission spectra.

The excited-state kinetics of the fluorescence of tyrosine in a de novo protein fibrillogenesis model was investigated as a potential tool for monitoring protein fibre formation and complexation with glucose (glycation). In stark contrast to insulin the time-resolved emission spectra (TRES) recorded over the period of 700 hours in buffered solutions of the model with and without glucose revealed no apparent changes in Tyr fluorescence responses. This indicates the stability of the model and provides a measurement-supported basis for its use as a reference material in fluorescence studies of protein aggregation.

Imaging Protein Fibers at the Nanoscale and In Situ.

Protein self-assembly offers a rich repertoire of tools and technologies. However, despite significant progress in this area, a deterministic measure of the phenomenon, which might lead to predictable relationships between protein components, assembly mechanisms, and ultimately function, is lacking. Often the challenge relates to the choice of the most informative and precise measurements that can link the chemistry of the building blocks with the resulting assembly, ideally in situ and in real time. Using the example of protein fibrillogenesis-a self-assembly process fundamental to nearly every aspect of biological organization, from viral assembly to tissue restoration-this chapter demonstrates how protein self-assembly can be visually and precisely measured while providing measurement protocols applicable to other self-assembly systems.

Linear and orthogonal peptide templating of silicified protein fibres.

Biomineralisation is essential for biology. Specialist proteins use peptide motifs that catalyse mineral deposition into nano-to-microscale inorganic materials. Unlike in native proteins, the motifs incorporated into self-assembled fibres can persistently propagate on the microscopic scale enabling empirically defined silica nanostructures. Herein we show that the two main modes of motif templating - linear and orthogonal - in self-assembling, fibre-forming peptide sequences effectively silicify protein fibres. We show that the mere charge and morphology of protein fibres are not sufficient for silica deposition, but it is the synergy between fibrillogenesis and silica-specific motifs regularly spaced in fibres that ensures silica templating, regardless of the relative orientation of the motifs.

Binary Encoding of Random Peptide Sequences for Selective and Differential Antimicrobial Mechanisms.

Binary encoding of peptide sequences into differential antimicrobial mechanisms is reported. Such sequences are random in composition, but controllable in chain length, are assembled from the same two amino acids, but differ in the stereochemistry of one. Regardless of chirality, the sequences lyse bacteria including the "superbugs" methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Sequences with the same chirality, so-called homochiral sequences, assemble into antimicrobial pores and form contiguous helices that are biologically promiscuous and hemolytic. By contrast, heterochiral sequences that lack such persistence selectively attack bacterial membranes without oligomerizing into visible pores. These results offer a mechanistic rationale for designing membrane-selective and sequence-independent antimicrobials.

Autonomously folded α-helical lockers promote RNAi.

RNAi is an indispensable research tool with a substantial therapeutic potential. However, the complete transition of the approach to an applied capability remains hampered due to poorly understood relationships between siRNA delivery and gene suppression. Here we propose that interfacial tertiary contacts between α-helices can regulate siRNA cytoplasmic delivery and RNAi. We introduce a rationale of helical amphipathic lockers that differentiates autonomously folded helices, which promote gene silencing, from helices folded with siRNA, which do not. Each of the helical designs can deliver siRNA into cells via energy-dependent endocytosis, while only autonomously folded helices with pre-locked hydrophobic interfaces were able to promote statistically appreciable gene silencing. We propose that it is the amphipathic locking of interfacing helices prior to binding to siRNA that enables RNAi. The rationale offers structurally balanced amphipathic scaffolds to advance the exploitation of functional RNAi.

A De Novo Virus-Like Topology for Synthetic Virions.

A de novo topology of virus-like assembly is reported. The design is a trifaceted coiled-coil peptide helix, which self-assembles into ultrasmall, monodisperse, anionic virus-like shells that encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, these shells share the same physical characteristics of viruses being anionic, nonaggregating, abundant, hollow, and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virus-like structures reported to date, both synthetic and native, with the ability to adapt and transfer small and large nucleic acids. The design thus offers a promising solution for engineering bespoke artificial viruses with desired functions.

Interfacial zippering-up of coiled-coil protein filaments.

Protein self-assembled materials find increasing use in medicine and nanotechnology. A challenge remains in our ability to tailor such materials at a given length scale. Here we report a de novo self-assembly topology which enables the engineering of filamentous protein nanostructures under morphological control. The rationale is exemplified by a ubiquitous self-assembly motif - an α-helical coiled-coil stagger. The stagger incorporates regularly spaced interfacial tryptophan residues, which allows it to zipper up into discrete filaments that bundle together without thickening by maturation. Using a combination of spectroscopy, microscopy, X-ray small-angle scattering and fibre diffraction methods we show that the precise positioning of tryptophan residues at the primary and secondary structure levels defines the extent of coiled-coil packing in resultant filaments. Applicable to other self-assembling systems, the rationale holds promise for the construction of advanced protein-based architectures and materials.

Peptide self-assembly for nanomaterials: the old new kid on the block.

Peptide self-assembly is an increasingly attractive tool for nanomaterials. Perfected in biology peptide self-assembling systems have impacted on nearly any conceivable nanomaterial type. However, with all the information available to us commercialisation of peptide materials remains in its infancy. In an attempt to better understand the reasons behind this shortcoming we categorise peptide self-assembled materials in relation to their non-peptide counterparts. A particular emphasis is placed on the versatility of peptide self-assembly in terms of modularity, responsiveness and functional diversity, which enables direct comparisons with more traditional material chemistries.