Effect of non-linear strain stiffening in eDAH and unjamming.

In cell clusters, the prominent factors at play encompass contractility-based enhanced tissue surface tension and cell unjamming transition. The former effect pertains to the boundary effect, while the latter constitutes a bulk effect. Both effects share outcomes of inducing significant elongation in cells. This elongation is so substantial that it surpasses the limits of linear elasticity, thereby giving rise to additional effects. To investigate these effects, we employ atomic force microscopy (AFM) to analyze how the mechanical properties of individual cells change under such considerable elongation. Our selection of cell lines includes MCF-10A, chosen for its pronounced demonstration of the extended differential adhesion hypothesis (eDAH), and MDA-MB-436, selected due to its manifestation of cell unjamming behavior. In the AFM analyses, we observe a common trend in both cases: as elongation increases, both cell lines exhibit strain stiffening. Notably, this effect is more prominent in MCF-10A compared to MDA-MB-436. Subsequently, we employ AFM on a dynamic range of 1-200 Hz to probe the mechanical characteristics of cell spheroids, focusing on both surface and bulk mechanics. Our findings align with the results from single cell investigations. Specifically, MCF-10A cells, characterized by strong contractile tissue tension, exhibit the greatest stiffness on their surface. Conversely, MDA-MB-436 cells, which experience significant elongation, showcase their highest stiffness within the bulk region. Consequently, the concept of single cell strain stiffening emerges as a crucial element in understanding the mechanics of multicellular spheroids (MCSs), even in the case of MDA-MB-436 cells, which are comparatively softer in nature.

Nonequilibrium Colloids: Temperature-Induced Bouquet Formation of Flower-like Micelles as a Time-Domain-Shifting Macromolecular Heat Alert.

Climate change requires enhanced autonomous temperature monitoring during logistics/transport. A cheap approach comprises the use of temperature-sensitive copolymers that undergo temperature-induced irreversible coagulation. The synthesis/characterization of pentablock copolymers (PBCP) starting from poloxamer PEO130-b-PPO44-b-PEO130 (poly(ethylene oxide)130-b-poly(propylene oxide)44-b-poly(ethylene oxide)130) and adding two terminal qPDMAEMA85 (quaternized poly[(2-dimethylamino)ethyl methacrylate]85) blocks is presented. Mixing of PBCP solutions with hexacyanoferrate(III)/ferricyanide solutions leads to a reduction of the decane/water interfacial tension accompanied by a co/self-assembly toward flower-like micelles in cold water because of the formation of an insoluble/hydrophobic qPDMAEMA/ferricyanide complex. In cold water, the PEO/PPO blocks provide colloidal stability over months. In hot water, the temperature-responsive PPO block is dehydrated, leading to a pronounced temperature dependence of the oil-water interfacial tension. In solution, the sticky PPO segments exposed at the micellar corona cause a colloidal clustering above a certain threshold temperature, which follows Smoluchowski-type kinetics. This coagulation remains for months even after cooling, indicating the presence of a kinetically trapped nonequilibrium state for at least one of the observed micellar structures. Therefore, the system memorizes a previous suffering of heat. This phenomenon is linked to an exchange of qPDMAEMA-blocks bridging the micellar cores after PPO-induced clustering. The addition of ferrous ions hampers the exchange, leading to the reversible coagulation of Prussian blue loaded micelles. Hence, the Fe2+ addition causes a shift from history monitoring to the sensing of the present temperature. Presumably, the system can be adapted for different temperatures in order to monitor transport and storage in a simple way. Hence, these polymeric "flowers" could contribute to preventing waste and sustaining the quality of goods (e.g., food) by temperature-induced bouquet formation, where an irreversible exchange of "tentacles" between the flowers stabilizes the bouquet at other temperatures as well.

Combining atomic force microscopy and fluorescence-based techniques to explore mechanical properties of naive and ischemia-affected brain regions in mice.

Knowledge of the brain's structure and function is essential for understanding processes in health and disease. Histochemical and fluorescence-based techniques have proven beneficial in characterizing brain regions and cellular compositions in pre-clinical research. Atomic force microscopy (AFM) has been introduced for mechanical tissue characterization, which may also help investigate pathophysiological aspects in disease-related models such as stroke. While combining AFM and fluorescence-based techniques, this study explored the mechanical properties of naive and ischemic brain regions in mice. Ischemia-affected regions were identified by the green signal of fluorescein isothiocyanate-conjugated albumin. A semi-automated protocol based on a brain atlas allowed regional allocations to the neocortex, striatum, thalamus, hypothalamus, hippocampus, and fiber tracts. Although AFM led to varying measurements, intra-individual analyses indicated a gradually increased tissue stiffness in the neocortex compared to subcortical areas, i.e., the striatum and fiber tracts. Regions affected by ischemia predominantly exhibited an increased tissue stiffness compared to those of the contra-lateral hemisphere, which might be related to cellular swelling. This study indicated intra-individual differences in mechanical properties among naive and ischemia-affected brain regions. The combination of AFM, semi-automated regional allocations, and fluorescence-based techniques thus qualifies for mechanical characterizations of the healthy and disease-affected brain in pre-clinical research.

Mechanical properties of murine hippocampal subregions investigated by atomic force microscopy and in vivo magnetic resonance elastography.

The hippocampus is a very heterogeneous brain structure with different mechanical properties reflecting its functional variety. In particular, adult neurogenesis in rodent hippocampus has been associated with specific viscoelastic properties in vivo and ex vivo. Here, we study the microscopic mechanical properties of hippocampal subregions using ex vivo atomic force microscopy (AFM) in correlation with the expression of GFP in presence of the nestin promoter, providing a marker of neurogenic activity. We further use magnetic resonance elastography (MRE) to investigate whether in vivo mechanical properties reveal similar spatial patterns, however, on a much coarser scale. AFM showed that tissue stiffness increases with increasing distance from the subgranular zone (p = 0.0069), and that stiffness is 39% lower in GFP than non-GFP regions (p = 0.0004). Consistently, MRE showed that dentate gyrus is, on average, softer than Ammon´s horn (shear wave speed = 3.2 ± 0.2 m/s versus 4.4 ± 0.3 m/s, p = 0.01) with another 3.4% decrease towards the subgranular zone (p = 0.0001). The marked reduction in stiffness measured by AFM in areas of high neurogenic activity is consistent with softer MRE values, indicating the sensitivity of macroscopic mechanical properties in vivo to micromechanical structures as formed by the neurogenic niche of the hippocampus.

The Cytoskeletal Elements MAP2 and NF-L Show Substantial Alterations in Different Stroke Models While Elevated Serum Levels Highlight Especially MAP2 as a Sensitive Biomarker in Stroke Patients.

In the setting of ischemic stroke, the neurofilament subunit NF-L and the microtubule-associated protein MAP2 have proven to be exceptionally ischemia-sensitive elements of the neuronal cytoskeleton. Since alterations of the cytoskeleton have been linked to the transition from reversible to irreversible tissue damage, the present study investigates underlying time- and region-specific alterations of NF-L and MAP2 in different animal models of focal cerebral ischemia. Although NF-L is increasingly established as a clinical stroke biomarker, MAP2 serum measurements after stroke are still lacking. Therefore, the present study further compares serum levels of MAP2 with NF-L in stroke patients. In the applied animal models, MAP2-related immunofluorescence intensities were decreased in ischemic areas, whereas the abundance of NF-L degradation products accounted for an increase of NF-L-related immunofluorescence intensity. Accordingly, Western blot analyses of ischemic areas revealed decreased protein levels of both MAP2 and NF-L. The cytoskeletal alterations are further reflected at an ultrastructural level as indicated by a significant reduction of detectable neurofilaments in cortical axons of ischemia-affected areas. Moreover, atomic force microscopy measurements confirmed altered mechanical properties as indicated by a decreased elastic strength in ischemia-affected tissue. In addition to the results from the animal models, stroke patients exhibited significantly elevated serum levels of MAP2, which increased with infarct size, whereas serum levels of NF-L did not differ significantly. Thus, MAP2 appears to be a more sensitive stroke biomarker than NF-L, especially for early neuronal damage. This perspective is strengthened by the results from the animal models, showing MAP2-related alterations at earlier time points compared to NF-L. The profound ischemia-induced alterations further qualify both cytoskeletal elements as promising targets for neuroprotective therapies.

Hierarchical drug release of pH-sensitive liposomes encapsulating aqueous two phase system.

As promising drug delivery vehicles, previous investigations of liposomes as carriers are primarily focused on insertion and modification of lipid membrane interfaces. The utility of the inner core seems to be overlooked. Herein, we developed pH-sensitive liposomes (PSLs) containing an aqueous two phase system (ATPS), and intriguingly discovered their hierarchical release under acidic stimuli. ATPS containing two polymers (poly(ethylene glycol) (PEG) and dextran) is homogeneous above phase transition temperature when producing ATPS-liposomes, and separated into PEG-rich phase and dextran-rich phase after cooling down to room temperature. The overall release time of ATPS-liposomes is divided into two stages and prolonged compared to simple aqueous liposomes. The unique release profile is due to the disproportional distribution of drugs in two phases. Doxorubicin (DOX) is loaded in the ATPS-liposomes, and their half maximum inhibition concentration on HeLa cells is 0.018 µmol L-1, which means 27.5 fold increase in inhibition efficiency over free DOX.

Direct measurement of surface charge distribution in phase separating supported lipid bilayers.

The local surface charge density of the cell membrane influences regulation and localization of membrane proteins. The local surface charge density could, until recently, not be measured directly under physiological conditions, and it was largely a hypothetical yet very important parameter. Here we use unsaturated lipids of a distinct charge (DOTAP, DOPC, and DOPG) and a neutral fully saturated lipid (DPPC) to create model membranes with phase separating domains of a defined charge. We then apply quantitative surface charge microscopy (QSCM) to investigate the local surface charge density; this is a technique based on a scanning ion conductance microscope (SICM) capable of measuring surface charge density with nanoscale lateral resolution. We are able to clearly distinguish lipid domains from charge and topography in all three model membranes. The measured surface charge densities furthermore reveal that disordered domains formed by charged lipids are in fact not only impure, but also incorporate uncharged saturated lipids. We estimate that at least 30% of disordered domains in DOPG : DPPC and DOTAP : DPPC will be DPPC. These ratios could present a limit for the formation of charged domains in lipid membranes.

Electroformation of double vesicles using an amplitude modulated electric field.

Double vesicles are a promising model to mimic eukaryotic cells, yet effective preparation methods with high yields and stable double vesicles are scarce. Previously reported electroformation methods were mainly based on sinusoidal AC fields. Using a combination of sinusoidal and amplitude modulated (AM) electric fields lipid double vesicles could be produced for the first time by a simple electroformation process. First lipid domes formed in a sinusoidal AC field. The domes grew into tubes during the subsequent application of an AM field. These tubes deformed into double vesicles to minimize their free energy in accordance with the area-difference-elasticity model. Two forces are involved to explain the mechanism behind tube formation. The pulling force (F) is responsible to drag the domes into tubular vesicles, but has to overcome a critical force (Fc). The most important parameters of the electrical field were explored systematically. In our work, a maximum yield for double vesicles of 63% was achieved. These vesicles proved to be stable for one week at least. Hence our method could provide a way to fabricate novel cell models.

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy.

Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.

A novel approach for mechanical tissue characterization indicates decreased elastic strength in brain areas affected by experimental thromboembolic stroke.

As treatment of ischemic stroke remains a challenge with respect to the failure of numerous neuroprotective attempts, there is an ongoing need for better understanding of pathophysiological mechanisms causing tissue damage. Although ischemic outcomes have been studied extensively at the cellular and molecular level using histological and biochemical methods, properties of ischemia-affected brain tissue with respect to mechanical integrity have not been addressed so far. As a novel approach, this study used fluorescence-based detection of regions affected by experimental thromboembolic stroke in combination with scanning force microscopy to examine mechanical alterations in selected rat brain areas. Twenty-five hours after onset of ischemia, a decreased elastic strength in the striatum as the region primarily affected by ischemia was found compared with the contralateral nonaffected hemisphere. Additional intrahemispheric analyses showed decreased elastic strength in the ischemic border zone compared with the more severely affected striatum. In conclusion, these data strongly indicate a critical alteration in mechanical tissue integrity caused by focal cerebral ischemia. Further, on the basis of data that have been obtained in relation to the ischemic border zone, a shell-like pattern of mechanical tissue damage was found in good accordance with the penumbra concept. These findings might enable the development of specific therapeutic interventions to protect affected areas from critical loss of mechanical integrity.

Causes of retrograde flow in fish keratocytes.

Confronting motile cells with obstacles doubling as force sensors we tested the limits of the driving actin and myosin machinery. We could directly measure the force necessary to stop actin polymerization as well as the force present in the retrograde actin flow. Combined with detailed measurements of the retrograde flow velocity and specific manipulation of actin and myosin we found that actin polymerization and myosin contractility are not enough to explain the cells behavior. We show that ever-present depolymerization forces, a direct entropic consequence of actin filament recycling, are sufficient to fill this gap, even under heavy loads.

Inherently slow and weak forward forces of neuronal growth cones measured by a drift-stabilized atomic force microscope.

Previous results have shown that glial cells provide a soft environment for the neurons of the mammalian central nervous system (CNS). This raises the question whether neurons are confined to the CNS and cannot wander off into more rigid tissues, such as brain capillary walls. We investigated the mechanical properties and force generation of extending mouse retinal ganglion cells and NG108-15 growth cones (GCs) using different atomic force microscope (AFM)-based methods. For the first time, to our knowledge, we were able to measure the forward pushing forces at the leading edge of outgrowing neuronal GCs with our drift-stabilized AFM. Our results demonstrate that these GCs have neither the required stability nor the ability to produce forces necessary to penetrate tissues that are at least an order of magnitude stiffer.