INMdok (Leibniz Institute for New Materials)
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Role of actin cytoskeleton in cargo delivery mediated by vertically aligned silicon nanotubes
Get PDFNanofabrication technologies have been recently applied to the development of engineered nano–bio interfaces for manipulating complex cellular processes. In particular, vertically configurated nanostructures such as nanoneedles (NNs) have been adopted for a variety of biological applications such as mechanotransduction, biosensing, and intracellular delivery. Despite their success in delivering a diverse range of biomolecules into cells, the mechanisms for NN-mediated cargo transport remain to be elucidated. Recent studies have suggested that cytoskeletal elements are involved in generating a tight and functional cell–NN interface that can influence cargo delivery. In this study, by inhibiting actin dynamics using two drugs—cytochalasin D (Cyto D) and jasplakinolide (Jas), we demonstrate that the actin cytoskeleton plays an important role in mRNA delivery mediated by silicon nanotubes (SiNTs). Specifically, actin inhibition 12 h before SiNT-cellular interfacing (pre-interface treatment) significantly dampens mRNA delivery (with efficiencies dropping to 17.2% for Cyto D and 33.1% for Jas) into mouse fibroblast GPE86 cells, compared to that of untreated controls (86.9%). However, actin inhibition initiated 2 h after the establishment of GPE86 cell–SiNT interface (post-interface treatment), has negligible impact on mRNA transfection, maintaining > 80% efficiency for both Cyto D and Jas treatment groups. The results contribute to understanding potential mechanisms involved in NN-mediated intracellular delivery, providing insights into strategic design of cell–nano interfacing under temporal control for improved effectiveness
Melt Electrowriting of Scaffolds with a Porosity Gradient to Mimic the Matrix Structure of the Human Trabecular Meshwork
Get PDFThe permeability of the Human Trabecular Meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases like glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues, and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 μm and fiber diameters of 10-12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa, and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffolds design, i.e., density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW to reconstruct complex morphological features of natural tissues.Competing Interest StatementThe authors have declared no competing interest
Nanoscale Faceting and Ligand Shell Structure Dominate the Self-Assembly of Non-Polar Nanoparticles into Superlattices
Get PDFSelf-assembly of nanoscale structures at liquid-solid interfaces occurs in a broad range of industrial processes and is found in various phenomena in nature. Conventional theory assumes spherical particles and homogeneous surfaces, but that model is oversimplified, and nanoscale in-situ observations are needed for a more complete understanding. We used liquid phase scanning transmission electron microscopy (LP-STEM) to examine the interactions that direct the self-assembly of superlattices formed by gold nanoparticles (AuNPs) in non-polar liquids. Varying the molecular coating of the substrate modulated short-ranged attraction and led to switching between a range of different geometric structures including hexagonal close-packed (hcp), simple hexagonal (sh), dodecahedral quasi-crystal (dqc), and body-centered cubic (bcc) lattices, as well as random distributions. Langevin dynamics simulations explain the experimental results in terms of the interplay between nanoparticle faceting, ligand shell structure, and substrate-NP-interactions
Strategy for optimizing experimental settings for studying low atomic number colloidal assemblies using liquid phase scanning transmission electron microscopy
Get PDFObserving processes of nanoscale materials of low atomic number is possible using liquid phase electron microscopy (LP-EM). However, the achievable spatial resolution (d) is limited by radiation damage. Here, we examine a strategy for optimizing LP-EM experiments based on an analytical model and experimental measurements, and develop a method for quantifying image quality at ultra low electron dose De using scanning transmission electron microscopy (STEM). As experimental test case we study the formation of a colloidal binary system containing 30-nm diameter SiO2 nanoparticles (SiONPs), and 100-nm diameter polystyrene microspheres (PMs). We show that annular dark field (DF) STEM is preferred over bright field (BF) STEM for practical reasons. Precise knowledge of the material's density is crucial for the calculations in order to match experimental data. To calculate the detectability of nano-objects in an image, the Rose criterion for single pixels is expanded to a model of the signal to noise ratio obtained for multiple pixels spanning the image of an object. Using optimized settings, it is possible to visualize the radiation-sensitive, hierarchical low-Z binary structures, and identify both components
P2-type layered high-entropy oxides as sodium-ion cathode materials
Get PDFP2-type layered oxides with the general Na-deficient composition Na x TMO2 (x < 1, TM: transition metal) are a promising class of cathode materials for sodium-ion batteries. The open Na+ transport pathways present in the structure lead to low diffusion barriers and enable high charge/discharge rates. However, a phase transition from P2 to O2 structure occurring above 4.2 V and metal dissolution at low potentials upon discharge results in rapid capacity degradation. In this work, we demonstrate the positive effect of configurational entropy on the stability of the crystal structure during battery operation. Three different compositions of layered P2-type oxides were synthesized by solid-state chemistry, Na0.67(Mn0.55Ni0.21Co0.24)O2, Na0.67(Mn0.45Ni0.18Co0.24Ti0.1Mg0.03)O2 and Na0.67(Mn0.45Ni0.18Co0.18Ti0.1Mg0.03Al0.04Fe0.02)O2 with low, medium and high configurational entropy, respectively. The high-entropy cathode material shows lower structural transformation and Mn dissolution upon cycling in a wide voltage range from 1.5 to 4.6 V. Advanced operando techniques and post-mortem analysis were used to probe the underlying reaction mechanism thoroughly. Overall, the high-entropy strategy is a promising route for improving the electrochemical performance of P2 layered oxide cathodes for advanced sodium-ion battery applications
Redox-triggerable firefly luciferin-bioinspired hydrogels as injectable and cell-encapsulating matrices
Get PDFStimuli-responsive hydrogels are smart materials that respond to variations caused by external stimuli and that are currently exploited for biomedical applications such as biosensing, drug delivery and tissue engineering. The development of stimuli-responsive hydrogels with defined user control is relevant to realize materials with advanced properties. Recently, our group reported firefly luciferin-inspired hydrogel matrices for 3D cell culture. This platform exhibited advantages like rapid gelation rate and tunability of mechanical and biological properties. However, this first molecular design did not allow fine control of the gelation onset, which restricts application as a cell-encapsulating matrice with injectable and processable properties. In this article, we endow the firefly luciferin-inspired hydrogels with redox-triggering capability, to overcome the limitations of the previous system and to widen its application range. We achieve this goal by introducing protected macromers as hydrogel polymeric precursors that can be activated in the presence of a mild reductant, to trigger gel formation in situ with a high degree of control. We demonstrate that the regulation of molecular parameters (e.g., structure of the protecting group, reductant type) and environmental parameters (e.g., pH, temperature) of the deprotection reaction can be exploited to modulate materials properties. This redox-triggerable system enables precise control over gelation onset and kinetics, thus facilitating its utilization as an injectable hydrogel without negatively impacting its cytocompatibility. Our findings expand the current toolkit of chemically-based stimuli-responsive hydrogels
Dielectric layers of hybrid Gold@Polystyrene Nanoparticle Inks
Get PDFA new type of hybrid dielectric based on nanoparticles with gold cores with diameters of 2.9-8.2 nm and covalently bound thiol-terminated polystyrene shells (Mn = 5000 Da and Mn = 11000 Da) is introduced. Particle dispersions were spin coated as dielectric films of thin film capacitors. The metal contents were 5-31 vol%, and the particles packed randomly or in face-centred-cubic superstructures, mainly depending on the polymer shell. Films with 9 vol% metal and 2.9 nm cores had dielectric constants of 98@1 Hz. Small angle X-ray scattering, transmission electron microscopy, and impedance spectroscopy indicate that classical random capacitor-resistor network models partially describe the hybrid materials. The covalently attached polymer shells enabled higher metal contents than in conventional nanocomposites without the risk of conductive breakdown. Dielectric properties depended on the metal content and the core size, but not on the network structure. The frequency-dependent dielectric polarization mainly takes place at the interfacial areas, but is not considered in the classical models. Smaller core sizes increased internal interfacial areas at comparable metal fractions by 46 %, resulting in 40 % larger dielectric constants in agreement with the Maxwell-Wagner-Sillars model. Inkjet-printed capacitors were prepared with a capacitance of 2.0±0.1 nF@10 kHz over an area of 0.79 mm² on rigid substrates; they retained their functionality over 3500 bending cycles on flexible substrates.Ein neuartiges hybrides Dielektrikum aus Nanopartikeln mit Goldkernen eines Durchmessers von 2,9-8,2 nm und kovalent gebundenen Thiol-terminierten Polystyrolhüllen (Mn = 5000 und 11000 Da) wird gezeigt. Aus den Partikeldispersionen wurden durch Rotationsbeschichten dielektrische Filme für Dünnschichtkondensatoren mit Metallgehalten von 5-31 vol% präpariert. Die Partikel ordneten sich je nach Polymerhülle in zufälligen oder kubisch-flächenzentrierten Strukturen an. Filme mit 9 vol% Metall und 2,9 nm großen Kernen hatten Dielektrizitätskonstanten von 98@1 Hz. Kleinwinkel-Röntgenstreuung, Transmissionselektronenmikroskopie und Impedanzspektroskopie deuten an, dass klassische zufallsbedingte Kondensator-Widerstand-Netzwerkmodelle die Materialien teilweise beschreiben. Die Polymerhüllen ermöglichten hohe Metallgehalte ohne das Risiko leitender Pfade. Die dielektrischen Eigenschaften hingen vom Metallgehalt und der Kerngröße ab, nicht aber von der Netzwerkstruktur. Die dielektrische Polarisation findet primär an den Grenzflächen statt und wird in den klassischen Modellen nicht berücksichtigt. Kleinere Kerne erhöhten die internen Grenzflächen bei ähnlichen Metallanteilen um 46 %, was zu 40 % größeren Dielektrizitätskonstanten gemäß dem Maxwell-Wagner-Sillars-Modell führte. Kondensatoren mit einer Kapazität von 2,0 ± 0,1 nF@10 kHz über eine Fläche von 0,79 mm² wurden mit Tintenstrahldruck auf starren Substraten hergestellt; sie blieben auf flexiblen Substraten über 3500 Biegezyklen intakt
Relationship between corrosion and nanoscale friction on a metallic glass
Get PDFMetallic glasses are promising materials for micro-devices, where corrosion and friction limit their effectiveness and durability. We investigated nanoscale friction on a metallic glass in corrosive solutions after different immersion times using atomic force microscopy to elucidate the influence of corrosion on nanoscale friction. The evolution of friction upon repeated scanning cycles on the corroded surfaces reveals a bilayer surface oxide film, where the outer layer is removed by the scanning tip. Friction and adhesion after different immersion times in different solutions allow to compare the physicochemical processes of surface dissolution at the interfaces of the two layers. The findings contribute to the understanding of mechanical contacts with metallic glasses in corrosive conditions by exploring the interrelation of microscopic corrosion mechanisms and nanoscale friction
Characterization of the Elasticity of CD4+ T Cells: An Approach Based on Peak Force Quantitative Nanomechanical Mapping
Get PDFCD4+ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4+ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive.With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation.Graphical abstract: Overview of the experimental workflow.Individual experimental steps are shown on the left, hands on and incubation times for each step are shown right
Amoeboid Cell Migration through Regular Arrays of Micropillars under Confinement
Get PDFMigrating cells often encounter a wide variety of topographic features—including the presence of obstacles—when navigating through crowded biological environments. Unravelling the impact of topography and crowding on the dynamics of cells is key to better understand many essential physiological processes such as the immune response. We study how migration and search efficiency of HL-60 cells differentiated into neutrophils in quasi two-dimensional environments are influenced by the lateral and vertical confinement and spatial arrangement of obstacles. A microfluidic device is designed to track the cells in confining geometries between two parallel plates with distance h, in which identical micropillars are arranged in regular pillar forests. We find that at each cell-pillar contact event, the cell spends a finite time near the pillar surface, which is independent of the height h and the interpillar spacing e. At low pillar density regime, the directional persistence of cells reduces with decreasing h or e, influencing their diffusivity and first-passage properties. The dynamics is strikingly different at high pillar density regime, where the cells are in simultaneous contact with more than one pillar; the cell velocity and persistence are distinctly higher compared to dilute pillar configurations with the same h. Our simulations reveal that the interplay between cell persistence and cell-pillar interactions can dramatically affect cell diffusivity and, thus, its first-passage properties.Competing Interest StatementThe authors have declared no competing interest