Composite Bioprinted Hydrogels Containing Porous Polymer Microparticles Provide Tailorable Mechanical Properties for 3D Cell Culture.

The mechanical and architectural properties of the three-dimensional (3D) tissue microenvironment can have large impacts on cellular behavior and phenotype, providing cells with specialized functions dependent on their location. This is especially apparent in macrophage biology where the function of tissue resident macrophages is highly specialized to their location. 3D bioprinting provides a convenient method of fabricating biomaterials that mimic specific tissue architectures. If these printable materials also possess tunable mechanical properties, they would be highly attractive for the study of macrophage behavior in different tissues. Currently, it is difficult to achieve mechanical tunability without sacrificing printability, scaffold porosity, and a loss in cell viability. Here, we have designed composite printable biomaterials composed of traditional hydrogels [nanofibrillar cellulose (cellulose) or methacrylated gelatin (gelMA)] mixed with porous polymeric high internal phase emulsion (polyHIPE) microparticles. By varying the ratio of polyHIPEs to hydrogel, we fabricate composite hydrogels that mimic the mechanical properties of the neural tissue (0.1-0.5 kPa), liver (1 kPa), lungs (5 kPa), and skin (10 kPa) while maintaining good levels of biocompatibility to a macrophage cell line.

Towards using 3D cellular cultures to model the activation and diverse functions of macrophages.

The advent of 3D cell culture technology promises to enhance understanding of cell biology within tissue microenvironments. Whilst traditional cell culturing methods have been a reliable tool for decades, they inadequately portray the complex environments in which cells inhabit in vivo. The need for better disease models has pushed the development of effective 3D cell models, providing more accurate drug screening assays. There has been great progress in developing 3D tissue models in fields such as cancer research and regenerative medicine, driven by desires to recreate the tumour microenvironment for the discovery of new chemotherapies, or development of artificial tissues or scaffolds for transplantation. Immunology is one field that lacks optimised 3D models and the biology of tissue resident immune cells such as macrophages has yet to be fully explored. This review aims to highlight the benefits of 3D cell culturing for greater understanding of macrophage biology. We review current knowledge of macrophage interactions with their tissue microenvironment and highlight the potential of 3D macrophage models in the development of more effective treatments for disease.

Amyloid Evolution: Antiparallel Replaced by Parallel.

Several atomic structures have now been found for micrometer-scale amyloid fibrils or elongated microcrystals using a range of methods, including NMR, electron microscopy, and X-ray crystallography, with parallel ß-sheet appearing as the most common secondary structure. The etiology of amyloid disease, however, indicates nanometer-scale assemblies of only tens of peptides as significant agents of cytotoxicity and contagion. By combining solution X-ray with molecular dynamics, we show that antiparallel structure dominates at the first stages of aggregation for a specific set of peptides, being replaced by parallel at large length scales only. This divergence in structure between small and large amyloid aggregates should inform future design of molecular therapeutics against nucleation or intercellular transmission of amyloid. Calculations and an overview from the literature argue that antiparallel order should be the first appearance of structure in many or most amyloid aggregation processes, regardless of the endpoint. Exceptions to this finding should exist, depending inevitably on the sequence and on solution conditions.

Self-assembling peptide and protein amyloids: from structure to tailored function in nanotechnology.

Self-assembled peptide and protein amyloid nanostructures have traditionally been considered only as pathological aggregates implicated in human neurodegenerative diseases. In more recent times, these nanostructures have found interesting applications as advanced materials in biomedicine, tissue engineering, renewable energy, environmental science, nanotechnology and material science, to name only a few fields. In all these applications, the final function depends on: (i) the specific mechanisms of protein aggregation, (ii) the hierarchical structure of the protein and peptide amyloids from the atomistic to mesoscopic length scales and (iii) the physical properties of the amyloids in the context of their surrounding environment (biological or artificial). In this review, we will discuss recent progress made in the field of functional and artificial amyloids and highlight connections between protein/peptide folding, unfolding and aggregation mechanisms, with the resulting amyloid structure and functionality. We also highlight current advances in the design and synthesis of amyloid-based biological and functional materials and identify new potential fields in which amyloid-based structures promise new breakthroughs.

Comparing Gene Silencing and Physiochemical Properties in siRNA Bound Cationic Star-Polymer Complexes.

The translation of siRNA into clinical therapies has been significantly delayed by issues surrounding the delivery of naked siRNA to target cells. Here we investigate siRNA delivery by cationic acrylic polymers developed by Reversible Addition-Fragmentation chain Transfer (RAFT) mediated free radical polymerization. We investigated cell uptake and gene silencing of a series of siRNA-star polymer complexes both in the presence and absence of a protein "corona". Using a multidisciplinary approach including quantitative nanoscale mechanical-atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis we have characterized the nanoscale morphology, stiffness, and surface charge of the complexes with and without the protein corona. This is one of the first examples of a comprehensive physiochemical analysis of siRNA-polymer complexes being performed alongside in vitro biological assays, allowing us to describe a set of desirable physical features of cationic polymer complexes that promote gene silencing. Multifaceted studies such as this will improve our understanding of structure-function relationships in nanotherapeutics, facilitating the rational design of polymer-mediated siRNA delivery systems for novel treatment strategies.

Microtubule-Binding R3 Fragment from Tau Self-Assembles into Giant Multistranded Amyloid Ribbons.

Tau protein and its fragments self-assemble into amyloid fibrils in the presence of polyanions, such as heparin. By combining microscopy, scattering, and spectroscopy techniques, we studied the aggregation of the 26-mer Tau-derived peptide alone, Tau(306-327), the third repeat fragment (R3) of the microtubule-binding domain. We show that: i) the sole Tau(306-327) can self-assemble into amyloid fibrils without the need of aggregation-promoting polyanions; ii) the resulting structures consist of surprisingly large, well-ordered 2D laminated flat ribbons, with a log-normal distribution of the lateral width, reaching the unprecedented lateral size of 350 nm and/or 45 individual protofilaments, that is, the largest amyloid laminated structures ever observed for Tau or any other amyloidogenic sequence. Our results provide insight into the molecular determinants of Tau aggregation and open new perspectives in the understanding of the assembly of amyloid fibrils and ß-sheet-based biomaterials.

Biomimetic topography and chemistry control cell attachment to amyloid fibrils.

Networks of nanoscale fibrous coatings made from self-assembled peptides are promising candidates for biomaterials that can promote the growth of mammalian cells. One particularly attractive feature is the possibility of adding biofunctional sequences to peptides to promote cell attachment. We deconvolute the topographic and chemical effects of nanoscale fibrils on cells by depositing a plasma polymer film on TTR1-based fibrils decorated with a range of cell adhesive chemistries (RGD and cycloRGDfK), producing a surface that retains the nanoscale fibrous topography of surface-bound fibrils but lacks the fibril surface chemistry. The surface topography was found to influence cell toxicity and spreading, and the fibril surface chemistry influenced cell attachment and spreading. This study highlights the importance of considering both the chemical and physical features of novel biomaterials and illustrates the use of plasma polymer deposition as a tool for examining the relationship between amyloid fibril structure and function.

ILQINS hexapeptide, identified in lysozyme left-handed helical ribbons and nanotubes, forms right-handed helical ribbons and crystals.

Amyloid fibrils are implicated in over 20 neurodegenerative diseases. The mechanisms of fibril structuring and formation are not only of medical and biological importance but are also relevant for material science and nanotechnologies due to the unique structural and physical properties of amyloids. We previously found that hen egg white lysozyme, homologous to the disease-related human lysozyme, can form left-handed giant ribbons, closing into nanotubes. By using matrix-assisted laser desorption ionization mass spectrometry analysis, we here identify a key component of such structures: the ILQINS hexapeptide. By combining atomic force microscopy and circular dichorism, we find that this fragment, synthesized by solid-phase peptide synthesis, also forms fibrillar structures in water at pH 2. However, all fibrillar structures formed possess an unexpected right-handed twist, a rare chirality within the corpus of amyloid experimental observations. We confirm by small- and wide-angle X-ray scattering and molecular dynamics simulations that these fibrils are composed of conventional left-handed ß-sheets, but that packing stresses between adjacent sheets create this twist of unusual handedness. We also show that the right-handed fibrils represent a metastable state toward ß-sheet-based microcrystals formation.

Engineered lysozyme amyloid fibril networks support cellular growth and spreading.

Fibrous networks assembled from synthetic peptides are promising candidates for biomimetic cell culture platforms and implantable biomaterials. The ability of the materials to reproduce physiological cell-matrix interactions is essential. However, the synthetic complexity of such systems limits their applications, thus alternative materials are desirable. Here, we design lysozyme derived amyloid fibril networks with controllable topographies, and perform a comprehensive study of the response of cultured fibroblast and epithelial cells. At high surface coverage a favorable increase in spreading and the generation of focal adhesions was observed, due to a combination of biomimetic chemistry and morphology. Their ease of synthesis, makes the nanoscale fibrils presented here ideal materials for future clinical applications whereby large volumes of biomimetic biomaterials are required. Furthermore, the surface chemistry of the fibrils is sufficient for the promotion of focal adhesions with cultured cells, eliminating the need for complex protocols for fibril decoration with bioactive moieties.

Nanotopographic surfaces with defined surface chemistries from amyloid fibril networks can control cell attachment.

We show for the first time the possibility of using networks of amyloid fibrils, adsorbed to solid supports and with plasma polymer coatings, for the fabrication of chemically homogeneous surfaces with well-defined nanoscale surface features reminiscent of the topography of the extracellular matrix. The robust nature of the fibrils allows them to withstand the plasma polymer deposition conditions used with no obvious deleterious effect, thus enabling the underlying fibril topography to be replicated at the polymer surface. This effect was seen despite the polymer coating thickness being an order of magnitude greater than the fibril network. The in vitro culture of fibroblast cells on these surfaces resulted in increased attachment and spreading compared to flat plasma polymer films with the same chemical composition. The demonstrated technique allows for the rapid and reproducible fabrication of substrates with nanoscale fibrous topography that we believe will have applications in the development of new biomaterials allowing, for example, the investigation of the effect of extracellular matrix mimicking nanoscale morphology on cellular phenotype.

Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection.

The complex shape and plasticity of cells is an intricate issue for the measurement of molecular diffusion in plasma membranes by fluorescence correlation spectroscopy (FCS). An important precondition for accurate diffusion measurements is a sufficient flatness of the membrane over the considered region and the absence of non-membrane-bound fluorescence diffusion. A method is presented to identify axial motion components caused by a non-ideal geometry of the membrane based on simultaneous measurement of the fluorescence emitted above and below the critical angle of the specimen/glass interface. Thereby, two detection volumes are generated that are laterally coincident, but differ in their axial penetration of the specimen. The similarity between the intensity tracks of the supercritical angle fluorescence (SAF) and the undercritical angle fluorescence (UAF) strongly depends on the membrane flatness and intracellular fluorescence, and can help to avoid sample-related artifacts in the diffusion measurement.

Lipid membrane-mediated assembly of the functional amyloid-forming peptide Somatostatin-14.

Membrane-mediated assembly has been well characterised for toxic amyloid species such as the amyloid-ß peptide implicated in Alzheimer's disease. However, little is known about the membrane-mediated assembly of functional-amyloid forming peptides, recently identified as a natural storage state for neuropeptide hormones in vivo. Here, we study the aggregation of somatostatin-14 (SST-14) co-incubated with model lipid membranes. Atomic force microscopy (AFM) studies confirmed that nanofibrils formed in the presence of various lipid membranes display reduced fibrillogenesis and promote the formation of non-fibrillar oligomers. Both circular dichroism (CD) and intrinsic tryptophan fluorescence studies confirmed interaction between the peptide and the lipid bilayer; this interaction appears to drive changes in membrane-mediated aggregation kinetics. We show that both the surface charge of the membrane and chain packing drive changes in the electrostatic and hydrophobic interactions between the peptide and the membrane, and hence the rate of assembly. The similarities in the effect of the lipid membrane on aggregation of functional amyloids and the more well studied toxic amyloids suggest strong aggregation modifying lipid bilayer interactions are a ubiquitous feature of all amyloid fibrils and highlight the need for further investigation as to why this leads to toxicity in some systems and not others.

Neurotoxic amyloidogenic peptides in the proteome of SARS-COV2: potential implications for neurological symptoms in COVID-19.

COVID-19 is primarily known as a respiratory disease caused by SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, severe headaches, and even stroke are reported in up to 30% of cases and can persist even after the infection is over (long COVID). These neurological symptoms are thought to be produced by the virus infecting the central nervous system, however we don't understand the molecular mechanisms triggering them. The neurological effects of COVID-19 share similarities to neurodegenerative diseases in which the presence of cytotoxic aggregated amyloid protein or peptides is a common feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we identified two peptides from the SARS-CoV-2 proteome that self-assemble into amyloid assemblies. Furthermore, these amyloids were shown to be highly toxic to neuronal cells. We suggest that cytotoxic aggregates of SARS-CoV-2 proteins may trigger neurological symptoms in COVID-19.

Mechanism of membrane interaction and disruption by α-synuclein.

Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-ß-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.

Photothermal release and recovery of mesenchymal stem cells from substrates functionalized with gold nanorods.

Mesenchymal stem cell therapies show great promise in regenerative medicine. However, to generate clinically relevant numbers of these stem cells, significant in vitro expansion of the cells is required before transplantation into the affected wound or defect. The current gold standard protocol for recovering in vitro cultured cells involves treatment with enzymes such as trypsin which can affect the cell phenotype and ability to interact with the environment. Alternative enzyme free methods of adherent cell recovery have been investigated, but none match the convenience and performance of enzymatic detachment. In this work we have developed a synthetically simple, low cost cell culture substrate functionalized with gold nanorods that can support cell proliferation and detachment. When these nanorods are irradiated with biocompatible low intensity near infrared radiation (785 nm, 560 mWcm-2) they generate localized surface plasmon resonance induced nanoscale heating effects which trigger detachment of adherent mesenchymal stem cells. Through simulations and thermometry experiments we show that this localized heating is concentrated at the cell-nanorod interface, and that the stem cells detached using this technique show either similar or improved multipotency, viability and ability to differentiate into clinically desirable osteo and adipocytes, compared to enzymatically harvested cells. This proof-of-principle work shows that photothermally mediated cell detachment is a promising method for recovering mesenchymal stem cells from in vitro culture substrates, and paves the way for further studies to scale up this process and facilitate its clinical translation. STATEMENT OF SIGNIFICANCE: New non-enzymatic methods of harvesting adherent cells without damaging or killing them are highly desirable in fields such as regenerative medicine. Here, we present a synthetically simple, non-toxic, infra-red induced method of harvesting mesenchymal stem cells from gold nanorod functionalized substrates. The detached cells retain their ability to differentiate into therapeutically valuable osteo and adipocytes. This work represents a significant improvement on similar cell harvesting studies due to: its simplicity; the use of clinically valuable stem cells as oppose to immortalized cell lines; and the extensive cellular characterization performed. Understanding, not just if cells live or die but how they proliferate and differentiate after photothermal detachment will be essential for the translation of this and similar techniques into commercial devices.

Molecular engineering of piezoelectricity in collagen-mimicking peptide assemblies.

Realization of a self-assembled, nontoxic and eco-friendly piezoelectric device with high-performance, sensitivity and reliability is highly desirable to complement conventional inorganic and polymer based materials. Hierarchically organized natural materials such as collagen have long been posited to exhibit electromechanical properties that could potentially be amplified via molecular engineering to produce technologically relevant piezoelectricity. Here, by using a simple, minimalistic, building block of collagen, we fabricate a peptide-based piezoelectric generator utilising a radically different helical arrangement of Phe-Phe-derived peptide, Pro-Phe-Phe and Hyp-Phe-Phe, based only on proteinogenic amino acids. The simple addition of a hydroxyl group increases the expected piezoelectric response by an order of magnitude (d35 = 27 pm V-1). The value is highest predicted to date in short natural peptides. We demonstrate tripeptide-based power generator that produces stable max current >50 nA and potential >1.2 V. Our results provide a promising device demonstration of computationally-guided molecular engineering of piezoelectricity in peptide nanotechnology.

Human neuropeptide substance P self-assembles into semi-flexible nanotubes that can be manipulated for nanotechnology.

Substance P neuropeptide is here reported to self-assemble into well-defined semi-flexible nanotubes. Using a blend of synchrotron small angle X-ray scattering, atomic force microscopy and other biophysical techniques, the natural peptide is shown to self-assemble into monodisperse 6 nm wide nanotubes, which can closely associate into nano-arrays with nematic properties. Using simple protocols, the nanotubes could be precipitated or mineralised while conserving their dimensions and core-shell morphology. Our discovery expands the small number of available monodisperse peptide nanotube systems for nanotechnology, beyond direct relevance to biologically functional peptide nanostructures since the substance P nanotubes are fundamentally different from typical amyloid fibrils.

Nanomechanical Properties and Phase Behavior of Phenylalanine Amyloid Ribbon Assemblies and Amorphous Self-Healing Hydrogels.

Phenylalanine was the minimalistic and first of numerous nonproteinaceous building blocks to be demonstrated to form amyloid-like fibrils. This unexpected organization of such a simple building block into canonical architecture, which was previously observed only with proteins and peptides, has numerous implications for medicine and supramolecular chemistry. However, the morphology of phenylalanine fibrils and their mechanical properties was never characterized in solutions. Here, using electron and atomic force microscopy, we analyze the morphological and mechanical properties of phenylalanine fibrils in both air and fluids. The fibrils demonstrate an exceptionally high Young's modulus (up to 30 GPa) and are found to be composed of intertwined protofilaments in a helical or twisted ribbon morphology. In addition, X-ray scattering experiments provide convincing evidence of an amyloidal cross-ß-like secondary structure within the nanoassemblies. Furthermore, increasing the phenylalanine concentration results in the formation of highly homogenous, noncrystalline, self-healing hydrogels that display storage and loss moduli significantly higher than similar noncovalently cross-linked biomolecular nanofibrillar scaffolds. These remarkably stiff nanofibrillar hydrogels can be harnessed for various technological and biomedical applications, such as self-healing, printable, structural, load-bearing 3D scaffolds. The properties of this simple but quite remarkable hydrogel open a possibility to utilize it in the biomaterial industry.

Nanomechanical mapping reveals localized stiffening of the basilar membrane after cochlear implantation.

Cochlear implantation leads to many structural changes within the cochlea which can impair residual hearing. In patients with preserved low-frequency hearing, a delayed hearing loss can occur weeks-to-years post-implantation. We explore whether stiffening of the basilar membrane (BM) may be a contributory factor in an animal model. Our objective is to map changes in morphology and Young's modulus of basal and apical areas of the BM after cochlear implantation, using quantitative nanomechanical atomic force microscopy (QNM-AFM) after cochlear implant surgery. Cochlear implantation was undertaken in the guinea pig, and the BM was harvested at four time-points: 1 day, 14 days, 28 days and 84 days post-implantation for QNM-AFM analysis. Auditory brainstem response thresholds were determined prior to implantation and termination. BM tissue showed altered morphology and a progressive increase in Young's modulus, mainly in the apex, over time after implantation. BM tissue from the cochlear base demonstrated areas of extreme stiffness which are likely due to micro-calcification on the BM. In conclusion, stiffening of the BM after cochlear implantation occurs over time, even at sites far apical to a cochlear implant.

Site-specific immobilization and micrometer and nanometer scale photopatterning of yellow fluorescent protein on glass surfaces.

A simple method is described for the site-specific attachment of yellow fluorescent protein (YFP) to glass surfaces on length scales ranging from tens of micrometers to ca. 200 nm. 3-Mercaptopropyl(triethoxy silane) is adsorbed onto a glass substrate and subsequently derivatized using a maleimide-functionalized oligomer of ethylene glycol. The resulting protein-resistant surface is patterned by exposure to UV light, causing photochemical degradation of the oligo(ethylene glycol) units to yield aldehyde groups in exposed regions. These are covalently bound to N-(5-amino-1-carboxypentyl)iminoacetic acid, yielding a nitrilotriacetic acid (NTA)-functionalized surface, which following complexation with Ni(2+), is coupled to His-tagged YFP. Using scanning near-field photolithography, in which a UV laser coupled to a scanning near-field optical microscope is utilized as the light source for photolithography, it is possible to fabricate lines of protein smaller than 200 nm, in which the biomolecules remain strongly optically active, facilitating the acquisition of diffraction-limited fluorescence images by confocal microscopy.