ABSTRACT Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

Abstract With the continuous advancement of microscopic imaging techniques, the motion of micro- and nanosized particles can be followed with increasing spatial and temporal accuracy. Analyzing the tracks of single particles enables one to address a broad range of scientific questions, e.g., the dynamics of active and passive (thermally driven) motion or the transport of particles in complex environments. While anisotropic motion, which is exhibited by the vast majority of biologically relevant particles (e.g., bacteria and viruses), has been amply studied in theoretical works, only little experimental data with single-particle resolution have been reported so far. In this study, fluorescence microscopy is used to follow the unconstrained motion of green-fluorescent protein (GFP)-labeled Salmonella enterica serovar Typhimurium (S. Typhimurium) in bulk. By decomposing the motion of individual bacteria into contributions parallel and perpendicular to the bacterial long axis, we introduce new scores that quantify the degree of motile activity and alignment with high throughput. We find that S. Typhimurium possesses a surprisingly broad spectrum of opening angles that range from full alignment of the bacterial long axis with the direction of translational motion to no alignment at all. Applied to mutants, in which motility-related proteins (the flagellins FljB and FliC) have been deleted, we find that wild-type (WT) and FliC-expressing S. Typhimurium exhibit very similar motility but differ significantly in the fraction of actively moving bacteria.

A recently published comment challenges the data analysis we employed to quantify nanomechanical properties in our force spectroscopy experiments. In the corresponding measurements, sub-µm-sized fluorescent beads are linked via nm-sized macromolecules ("linker") to the bottom of a microfluidic channel. Application of a flow through the channel generates a well-defined shear force acting on the bead, which deforms the linker and can be followed with nm spatial and sub-pN force resolution and high data throughput using optical microscopy. The comment challenges our analysis and proposes a different model for the analysis of our data. Based on this model, the authors claim that for certain measurements, the loading force was underestimated by a factor of up to 30. In our response, we discuss the rationale behind our model in detail and provide experimental evidence that supports our rationale. This data shows that the proposed model is oversimplified in the sense that important restoring forces have not been taken into account. As a consequence, the proposed model does not reflect all features of our data and is therefore not applicable to our experimental setting. We therefore dismiss the claim that we underestimated the loading force by orders of magnitude.

Proper regulation of airway surface layer (ASL) is essential for effective mucociliary clearance (MCC) in healthy airways. ASL depletion due to deficient cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion/fluid secretion plays an important role in the pathogenesis of mucociliary dysfunction and chronic muco-obstructive lung disease in patients with cystic fibrosis (CF). Quantitative measurement of ASL height by confocal fluorescence microscopy following addition of fluorescent dye has contributed important insight into the (dys)regulation of ASL in health and disease. Here, we present a novel method enabling studies of ASL regulation that does not require the addition of dye by using reflected light by confocal microscopy of primary airway epithelial cultures grown at air-liquid interface (ALI). After apical volume addition to primary tracheal mouse cultures, confocal reflection microscopy yielded comparable ASL height as confocal fluorescence microscopy on cultures of wild-type mice, and was sensitive to detect ASL depletion on cultures of βENaC-Tg mice. Under unperturbed conditions, ASL determined by confocal reflection microscopy was significantly higher in wild-type and βENaC-Tg mice compared to values obtained by confocal fluorescence microscopy. Studies in normal and CF primary human airway epithelial cultures showed that confocal reflection microscopy was sensitive to detect effects of low temperature rescue and pharmacological modulation including improvement of CFTR function by VX-809 and VX-770 in cultures from CF patients with the F508del mutation. Our results support confocal reflection microscopy as a novel sensitive technique for quantitative studies of ASL regulation and response to therapeutic intervention under near-physiological conditions that may be applicable for studies of (patho)physiology and drug screens in healthy and CF airways.

Mucus is a complex hydrogel that acts as a defensive and protective barrier in various parts of the human body. The rise in the level of viral infections has underscored the importance of advancing research into mucus-mimicking hydrogels for the efficient design of antiviral agents. Herein, we demonstrate the gram-scale synthesis of biocompatible, lignin-based virus-binding inhibitors that reduce waste and ensure long-term availability. The lignin-based inhibitors are equipped with sulfate moieties, which are known binding partners for many viruses, including SARS-CoV-2 and herpes viruses. In addition, cross-linking the synthesized inhibitors yielded hydrogels that mimicked native mucus concerning surface functionality and rheology. The degree of sulfation exhibits a very strong impact on the mesh size distribution of the hydrogels, which provides a new means to fine-tune the steric and electrostatic contributions of the virus-hydrogel interaction. This feature strongly impacts the sequestration capability of the lignin-based hydrogels, which is demonstrated by infection inhibition assays involving human herpes simplex virus 1, influenza A viruses, and the bacterium Escherichia coli (E. coli). These measurements showed a reduction in plaque-forming units (HSV-1) and colony-forming units (E. coli) by more than 4 orders of magnitude, indicating the potent inhibition by the lignin-based hydrogels.

When analyzing the individual positional dynamics of an ensemble of moving objects, the extracted parameters that characterize the motion of individual objects, such as the mean-squared instantaneous velocity or the diffusivity, exhibit a spread that is due to the convolution of three different effects: i) Motion stochasticity, caused by the fluctuating environment and enhanced by limited observation time, ii) measurement errors that depend on details of the detection technique, and iii) the intrinsic parameter variance that characterizes differences between individual objects, the quantity of ultimate interest. We develop the theoretical framework to separate these effects using the generalized Langevin equation (GLE), which constitutes the most general description of active and passive dynamics, as it derives from the general underlying many-body Hamiltonian for the studied system without approximations. We apply our methodology to determine intrinsic cell-to-cell differences of living human breast-cancer cells, algae cells and, as a benchmark, size differences of passively moving polystyrene beads in water. We find algae and human breast-cancer cells to exhibit significant individual differences, reflected by the spreading of the intrinsic mean-squared instantaneous velocity over two orders of magnitude, which is remarkable in light of the genetic homogeneity of the investigated breast-cancer cells and highlights their phenotypical diversity. Quantification of the intrinsic variance of single-cell properties is relevant for infection biology, ecology and medicine and opens up new possibilities to estimate population heterogeneity on the single-organism level in a non-destructive manner. Our framework is not limited to motility properties but can be readily applied to general experimental time-series data.

Mucus is a complex hydrogel acting as a defensive and protective barrier in various parts of the human body. The structure and composition of mucus play an important role in maintaining barrier properties by acting as a filter for the diffusion of biomolecules and pathogens. The rise in viral infections has underscored the importance of advancing research into mucus-mimicking hydrogels for the efficient design of antiviral agents. However, the performance of an antiviral strategy should not only be assessed based on its efficacy in inhibiting infections but also based on its sustainability. Herein, we demonstrate the gram-scale synthesis of biocompatible, lignin-based virus-binding inhibitors that reduce waste and ensure long-term availability. The lignin-based inhibitors were equipped with sulfate moieties, which are known binding partners for many viruses including SARS-CoV-2 and herpes viruses. In addition, crosslinking the synthesized inhibitors yielded hydrogels that mimicked native mucus with respect to surface functionality and rheology. It is found that the degree of sulfation has a very strong impact on the mesh size distribution of the hydrogels, which provides a new means to fine-tune steric and electrostatic contributions of the virus-hydrogel interaction. This feature strongly impacts the sequestration capability of the lignin-based hydrogels, which is demonstrated by infection inhibition assays involving human herpes simplex virus-1, influenza A viruses, and the bacterium Escherichia coli (E. coli). For HSV-1 and E. coli, these measurements showed a reduction in plaque (HSV-1) and colony-forming units (E. coli) by more than 4 orders of magnitude, indicating potent inhibition by the lignin-based hydrogels. Taken together, the sulfated lignin hydrogel is an excellent scaffold for large-scale synthesis of sustainable, biocompatible, and highly efficient pathogen-binding inhibitors.

Die Bindung von Viren an Wirtszellen sowie die anschließende Membranfusion sind entscheidende Schritte im Verlauf einer Virusinfektion. Diese Prozesse werden durch virale Proteine vermittelt, die spezifisch an Zelloberflächenrezeptoren binden und die Fusion der viralen mit der zellulären Membran ermöglichen. Um diese Mechanismen für die Entwicklung neuer antiviraler Strategien gezielt angehen zu können, sind Methoden erforderlich, die Virus‐Membran‐Interaktionen unter in‐situ‐Bedingungen untersuchen und mechanistische Einblicke auf molekularer Ebene liefern. In dieser Arbeit stellen wir den Einsatz der oberflächenverstärkten Infrarot‐Absorptionsspektroskopie (SEIRA, engl. Surface‐Enhanced Infrared Absorption ) in Kombination mit verankerten Doppelschichtlipidmembranen (tBLMs, engl. tethered bilayer lipid membranes ) zur markierungsfreien Detektion von Virus‐Membran‐Interaktionen vor, wobei das Influenza‐A/X‐31‐Virus (IAV) als Modell dient. Unter Ausnutzung der nanometerskaligen Oberflächensensitivität der SEIRA‐Spektroskopie detektieren wir den Schwingungsfingerabdruck des Hämagglutinin‐(HA)‐Glykoproteins des IAV bei der spezifischen Bindung an die Sialinsäuren des Gangliosids GD1a in der tBLM, welche die Wirtszellmembran nachbildet. Durch eine pH‐Änderung induzieren wir die virale Fusion und identifizieren strukturelle Veränderungen im HA während seiner Interaktion mit dem Wirtsmembranmodell. Zudem ermöglicht der Einsatz deuterierter Lipide in der tBLM die Nutzung des Schwingungsisotopeneffekts, wodurch Virus‐ und Modellmembran voneinander unterschieden und Lipidmischvorgänge verfolgt werden können. Diese Methode ermöglicht neue spektroskopische Untersuchungen der Funktion und Inhibition viraler Proteine innerhalb intakter Viruspartikel.

Viral binding and membrane fusion are essential steps in viral infection, mediated by viral proteins that bind to host cell receptors and facilitate the fusion between viral and host membranes. Targeting these steps for the development of new antiviral strategies requires methods that enable investigating virus-membrane interactions under in-situ conditions, while providing mechanistic insights on a molecular level. Here, we demonstrate the use of surface-enhanced infrared absorption (SEIRA) spectroscopy combined with tethered bilayer lipid membranes (tBLMs) for the label-free detection of virus-membrane interactions, using the Influenza A/X-31 virus (IAV) as a model. Exploiting the nanometer-scale surface-sensitivity of SEIRA, we detect the vibrational fingerprint of IAV's hemagglutinin (HA) glycoprotein, as it specifically binds to sialic acid receptors of the ganglioside GD1a in the tBLM, mimicking the host membrane. Triggering viral fusion via a pH change, we identify structural changes of HA engaging with the host membrane model. Moreover, by constructing the tBLM from deuterated lipids, we utilize the vibrational isotope effect and distinguish between viral and model membrane, providing a basis to track lipid mixing. This approach establishes a powerful tool for spectroscopic studies of the function and inhibition of viral proteins, while still embedded in intact virus particles.

Glycosylated RNA (glycoRNA) has recently emerged as a novel constituent of the glycocalyx on cell surfaces, yet its biological functions remain largely unexplored. In this report, we present the first analysis of glycoRNA expression and functionality in alveolar epithelial cells. To this end, we optimized new techniques for the detection of glycoRNA on living cell surfaces and in cell membrane-associated RNA samples through in-gel imaging after labeling with fluorescent dye conjugates. Specifically, we used conjugation of Cy5-hydrazide after mild oxidation with sodium periodate for detection of total cell surface sialoglycoRNA. Conjugation of dibenzocyclooctyne-sulfo-Cy5 in cells fed with tetraacetylated N-azidoacetyl-mannosamine or 6-azido-L-fucose detected de novo-formed sialoglycoRNA or fucoglycoRNA, respectively. Finally, biotinylated lectins in combination with infrared dye-conjugated streptavidin were used to differentiate between specific glycosidic linkages. Comparisons across primary alveolar epithelial cells and different alveolar epithelial-like cell lines revealed a cell-type-specific variation in glycoRNA abundance. Treatment of primary alveolar epithelial cells with an RNase cocktail reduced epithelial surface glycoRNA and was associated with a reduction in transepithelial electrical resistance and influenza A viral particle abundance. As such, the present work identifies glycoRNA as a novel component of the alveolar epithelial glycocalyx with potential relevance in epithelial barrier regulation and viral infection.

Cytochrome c oxidase (CcO) is a transmembrane protein and terminal oxidase in the respiratory electron transfer chain of many bacteria and in the mitochondria of eukaryotic cells. In this position, CcO receives electrons, which were generated during cellular respiration, and uses these electrons to reduce dioxygen to water, thereby acting as an electron sink in the respiratory chain. As electrons and protons are taken up from opposite sides of the membrane, the enzymatic activity of CcO contributes to the generation of an electrochemical gradient acting across the membrane hosting the respiratory chain proteins, which drives the synthesis of ATP. Due to its importance in bioenergetics, CcO is subject of intense studies for decades. Nevertheless, hardly any information is currently available regarding the proton uptake rate of CcO, especially regarding the impact of the electrochemical gradient on this rate. In this study, a single-proteoliposome assay is employed to follow proton uptake of individual CcOs from Rhodobacter sphaeroides operating against the action of a well-defined pH gradient. Our measurements reveal that proton uptake is not affected by small pH gradients (< 0.7 pH units) and decays exponentially for larger pH gradients. Furthermore, a linear dependence of substrate concentration on the proton uptake rate is observed over more than 3 orders of magnitude. The obtained scaling laws are surprisingly simple, considering the fact that 4 protons have to be taken up to reduce dioxygen to water, and are discussed in terms of the sequential uptake of protons and electrons occurring during CcOs catalytic cycle.

Blood vessel formation relies on biochemical and mechanical signals, particularly during sprouting angiogenesis when endothelial tip cells (TCs) guide sprouting through filopodia formation. The contribution of BMP receptors in defining tip-cell characteristics is poorly understood. Our study combines genetic, biochemical, and molecular methods together with 3D traction force microscopy, which reveals an essential role of BMPR2 for actin-driven filopodia formation and mechanical properties of endothelial cells (ECs). Targeting of Bmpr2 reduced sprouting angiogenesis in zebrafish and BMPR2-deficient human ECs formed fewer filopodia, affecting cell migration and actomyosin localization. Spheroid assays revealed a reduced sprouting of BMPR2-deficient ECs in fibrin gels. Even more strikingly, in mosaic spheroids, BMPR2-deficient ECs failed to acquire tip-cell positions. Yet, 3D traction force microscopy revealed that these distinct cell behaviors of BMPR2-deficient tip cells cannot be explained by differences in force-induced matrix deformations, even though these cells adopted distinct cone-shaped morphologies. Notably, BMPR2 positively regulates local CDC42 activity at the plasma membrane to promote filopodia formation. Our findings reveal that BMPR2 functions as a nexus integrating biochemical and biomechanical processes crucial for TCs during angiogenesis.

We developed a three-dimensional (3D) polyglycerol-poly(ethylene glycol)-based hydrogel as a new biosensing matrix for affinity analysis by surface plasmon resonance to enable a high loading of ligands for small molecule analysis while lacking a carbohydrate structure to reduce nonspecific binding. The hydrogel was synthesized by cross-linking a polyglycerol functionalized with carboxylate and maleimide groups with a dithiolated poly(ethylene glycol) by thiol-click chemistry. We demonstrated that the hydrogel coating enabled a high immobilization capacity of biomolecules and led to less nonspecific binding. Here, the degree of loading with carbonic anhydrase II and the resulting binding signal of acetazolamide were increased by a factor of 5 compared to standard CMD sensors (CM5), and the loading was comparable to CMD sensors specialized for maximum loading (CM7). This high loading capacity, combined with the reduced nonspecific binding due to the missing carbohydrate structure, presents an innovative matrix for a broad application range of surface plasmon resonance (SPR) experiments since no current commercial SPR biosensor combines these two key features.

Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad spectrum of viruses. Here we developed a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ~10 % repeating units of activated disulfide as cross-linking sites. Cryo-EM analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as HSV-1 and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched PEG-thiol as cross-linkers. Single particle tracking microrheology, EPR and Cryo-SEM were used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. a transwell assay was used to investigate the hydrogel’s protective properties against viral infection against HSV-1. Live-cell microscopy confirmed that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, our novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as a models for disulfide-rich airway mucus research, and as biomaterials.

Mucus forms the first defense line of human lungs, and as such hampers the efficient delivery of therapeutics to the underlying epithelium. This holds particularly true for genetic cargo such as CRISPR-based gene editing tools which cannot readily surmount the mucosal barrier. While lipid nanoparticles (LNPs) emerge as versatile non-viral gene delivery systems that can help overcome the delivery challenge, many knowledge gaps remain, especially for diseased states such as cystic fibrosis (CF). This study provides fundamental insights into Cas9 mRNA or ribonucleoprotein-loaded LNP-mucus interactions in healthy and diseased states by assessing the impact of the genetic cargo, mucin sialylation, mucin concentration, ionic strength, pH, and polyethylene glycol (PEG) concentration and nature on LNP diffusivity leveraging experimental approaches and Brownian dynamics (BD) simulations. Taken together, this study identifies key mucus and LNP characteristics that are critical to enabling a rational LNP design for transmucosal delivery.

We present a method to differentiate organisms solely by their motion based on the generalized Langevin equation (GLE) and use it to distinguish two different swimming modes of strongly confined unicellular microalgae Chlamydomonas reinhardtii. The GLE is a general model for active or passive motion of organisms and particles that can be derived from a time-dependent general many-body Hamiltonian and in particular includes non-Markovian effects (i.e., the trajectory memory of its past). We extract all GLE parameters from individual cell trajectories and perform an unbiased cluster analysis to group them into different classes. For the specific cell population employed in the experiments, the GLE-based assignment into the two different swimming modes works perfectly, as checked by control experiments. The classification and sorting of single cells and organisms is important in different areas; our method, which is based on motion trajectories, offers wide-ranging applications in biology and medicine.

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry.

Several techniques have been established to quantify the mechanicals of single molecules. However, most of them show only limited capabilities of parallelizing the measurement by performing many individual measurements simultaneously. Herein, a microfluidics-based single-molecule force spectroscopy method, which achieves sub-nanometer spatial resolution and sub-piconewton sensitivity and is capable of simultaneously quantifying hundreds of single-molecule targets in parallel, is presented. It relies on a combination of total internal reflection microscopy and microfluidics, in which monodisperse fluorescent beads are immobilized on the bottom of a microfluidic channel by macromolecular linkers. Application of a flow generates a well-defined shear force acting on the beads, whereas the nanomechanical linker response is quantified based on the force-induced displacement of individual beads. To handle the high amount of data generated, a cluster analysis which is capable of a semi-automatic identification of measurement artifacts and molecular populations is implemented. The method is validated by probing the mechanical response polyethylene glycol linkers and binding strength of biotin-NeutrAvidin complexes. Two energy barriers (at 3 and 5.7 Å, respectively) in the biotin-NeutrAvidin interaction are resolved and the unfolding behavior of talin's rod domain R3 in the force range between 1 to ≈10 pN is probed.

Heparan sulfate (HS) is a highly sulfated polysaccharide on the surface of mammalian cells and in the extracellular matrix and has been found to be important for virus binding and infection. In this work, we designed synthetic hydrogels with viral binding and deactivation activities through the postfunctionalization of an HS-mimicking polyelectrolyte and alkyl chains. Three polyglycerol-based hydrogels were prepared as substrates and postfunctionalized by sulfated linear polyglycerol (lPGS) via thiol-ene click reaction. The viral binding properties were studied using herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV). The effect of hydrogel types and molecular weight (Mw) of conjugated lPGS on viral binding properties was also assessed, and promising binding activities were observed in all lPGS-functionalized samples. Further coupling of 11 carbons long alkyl chains to the hydrogel revealed virucidal properties caused by destruction of the viral envelope, as shown by atomic force microscopy (AFM) imaging.