Exploring Mechanisms of Lipid Nanoparticle-Mucus Interactions in Healthy and Cystic Fibrosis Conditions.

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.

Human disease models in drug development.

Biomedical research is undergoing a paradigm shift towards approaches centred on human disease models owing to the notoriously high failure rates of the current drug development process. Major drivers for this transition are the limitations of animal models, which, despite remaining the gold standard in basic and preclinical research, suffer from interspecies differences and poor prediction of human physiological and pathological conditions. To bridge this translational gap, bioengineered human disease models with high clinical mimicry are being developed. In this Review, we discuss preclinical and clinical studies that benefited from these models, focusing on organoids, bioengineered tissue models and organs-on-chips. Furthermore, we provide a high-level design framework to facilitate clinical translation and accelerate drug development using bioengineered human disease models.

Zasp52 strengthens whole embryo tissue integrity through supracellular actomyosin networks.

During morphogenesis, large-scale changes of tissue primordia are coordinated across an embryo. In Drosophila, several tissue primordia and embryonic regions are bordered or encircled by supracellular actomyosin cables, junctional actomyosin enrichments networked between many neighbouring cells. We show that the single Drosophila Alp/Enigma-family protein Zasp52, which is most prominently found in Z-discs of muscles, is a component of many supracellular actomyosin structures during embryogenesis, including the ventral midline and the boundary of the salivary gland placode. We reveal that Zasp52 contains within its central coiled-coil region a type of actin-binding motif usually found in CapZbeta proteins, and this domain displays actin-binding activity. Using endogenously-tagged lines, we identify that Zasp52 interacts with junctional components, including APC2, Polychaetoid and Sidekick, and actomyosin regulators. Analysis of zasp52 mutant embryos reveals that the severity of the embryonic defects observed scales inversely with the amount of functional protein left. Large tissue deformations occur where actomyosin cables are found during embryogenesis, and in vivo and in silico analyses suggest a model whereby supracellular Zasp52-containing cables aid to insulate morphogenetic changes from one another.

Poroelastic modelling reveals the cooperation between two mechanisms for albuminuria.

Albuminuria occurs when albumin leaks abnormally into the urine. Its mechanism remains unclear. A gel-compression hypothesis attributes the glomerular barrier to compression of the glomerular basement membrane (GBM) as a gel layer. Loss of podocyte foot processes would allow the gel layer to expand circumferentially, enlarge its pores and leak albumin into the urine. To test this hypothesis, we develop a poroelastic model of the GBM. It predicts GBM compression in healthy glomerulus and GBM expansion in the diseased state, essentially confirming the hypothesis. However, by itself, the gel compression and expansion mechanism fails to account for two features of albuminuria: the reduction in filtration flux and the thickening of the GBM. A second mechanism, the constriction of flow area at the slit diaphragm downstream of the GBM, must be included. The cooperation between the two mechanisms produces the amount of increase in GBM porosity expected in vivo in a mutant mouse model, and also captures the two in vivo features of reduced filtration flux and increased GBM thickness. Finally, the model supports the idea that in the healthy glomerulus, gel compression may help maintain a roughly constant filtration flux under varying filtration pressure.

A similarity scaling approach for organ-on-chip devices.

Organ-on-chip devices (OoCs) provide more nuanced insights into (patho)physiological processes of the human body than static tissue models, and are currently the most promising approach to emulating human (patho)physiology in vitro. OoC designs vary greatly and questions remain as to how to maximize biomimicry and clinical translatability of the in vitro findings. Scaling is critical, yet has largely been ad hoc, consisting in matching one or a few variables between the OoC and the target organ. This has limited the predictive value of OoCs. Here, we propose a systematic approach based on the principle of similitude widely used in the physical sciences, and present three case studies from the recent literature to demonstrate how the approach works. A lung-on-a-chip and a liver-on-a-chip both satisfied important similarity criteria, and therefore yielded results that were in good agreement with clinical data. A gut-liver system failed to satisfy a key criterion of kinematic similarity, and yielded unphysiological pharmacokinetic responses in vitro. The similarity scaling approach promises to improve markedly the design and operation of organ- and human-on-chip devices.

A mechanical test of the tenertaxis hypothesis for leukocyte diapedesis.

As part of the immune response, leukocytes can directly transmigrate through the body of endothelial cells or through the gap between adjacent endothelial cells. These are known, respectively, as the transcellular and paracellular route of diapedesis. What determines the usage of one route over the other is unclear. A recently proposed tenertaxis hypothesis claims that leukocytes choose the path with less mechanical resistance against leukocyte protrusions. We examined this hypothesis using numerical simulation of the mechanical resistance during paracellular and transcellular protrusions. By using parameters based on human lung endothelium, our results show that the required force to breach the endothelium through the transcellular route is greater than paracellular route, in agreement with experiments. Moreover, experiments have demonstrated that manipulation of the relative strength between the two routes can make the transcellular route preferable. Our simulations have demonstrated this reversal and thus tentatively confirmed the hypothesis of tenertaxis.

A three-dimensional vertex model forDrosophilasalivary gland invagination.

During epithelial morphogenesis, force generation at the cellular level not only causes cell deformation, but may also produce coordinated cell movement and rearrangement on the tissue level. In this paper, we use a novel three-dimensional vertex model to explore the roles of cellular forces during the formation of the salivary gland in theDrosophilaembryo. Representing the placode as an epithelial sheet of initially columnar cells, we focus on the spatial and temporal patterning of contractile forces due to three actomyosin pools: the apicomedial actomyosin in the pit of the placode, junctional actomyosin arcs outside the pit, and a supracellular actomyosin cable along the circumference of the placode. In anin silico'wild type' model, these pools are activated at different times according to experimental data. To identify the role of each myosin pool, we have also simulated variousin silico'mutants' in which only one or two of the myosin pools are activated. We find that the apicomedial myosin initiates a small dimple in the pit, but this is not essential for the overall invagination of the placode. The myosin arcs are the main driver of invagination and are responsible for the internalization of the apical surface. The circumferential actomyosin cable acts to constrict the opening of the developing tube, and is responsible for forming a properly shaped lumen. Cell intercalation tends to facilitate the invagination, but the geometric constraints of our model only allow a small number of intercalations, and their effect is minor. The placode invagination predicted by the model is in general agreement with experimental observations. It confirms some features of the current 'belt-and-braces' model for the process, and provides new insights on the separate roles of the various myosin pools and their spatio-temporal coordination.

Phase-field model for elastocapillary flows of liquid crystals.

We propose a phase-field model to study interfacial flows of nematic liquid crystals that couple the capillary forces on the interface with the elastic stresses in the nematic phase. The theoretical model has two key ingredients: A tensor order parameter that provides a consistent description of the molecular and distortional elasticity, and a phase-field formalism that accurately represents the interfacial tension and the nematic anchoring stress by approximating a sharp-interface limit. Using this model, we carry out finite-element simulations of drop retraction in a surrounding fluid, with either component being nematic. The results are summarized by eight representative steady-state solutions in planar and axisymmetric geometries, each featuring a distinct configuration for the drop and the defects. The dynamics is dominated by the competition between the interfacial tension and the distortional elasticity in the nematic phase, mediated by the anchoring condition on the drop surface. As consequences of this competition, the steady-state drop deformation and the clearance between the defects and the drop surface both depend linearly on the elastocapillary number.

Tear-film breakup: The role of membrane-associated mucin polymers.

Mucin polymers in the tear film protect the corneal surface from pathogens and modulate the tear-film flow characteristics. Recent studies have suggested a relationship between the loss of membrane-associated mucins and premature rupture of the tear film in various eye diseases. This work aims to elucidate the hydrodynamic mechanisms by which loss of membrane-associated mucins causes premature tear-film rupture. We model the bulk of the tear film as a Newtonian fluid in a two-dimensional periodic domain, and the lipid layer at the air-tear interface as insoluble surfactants. Gradual loss of membrane-associated mucins produces growing areas of exposed cornea in direct contact with the tear fluid. We represent the hydrodynamic consequences of this morphological change through two mechanisms: an increased van der Waals attraction due to loss of wettability on the exposed area, and a change of boundary condition from an effective negative slip on the mucin-covered areas to the no-slip condition on exposed cornea. Finite-element computations, with an arbitrary Lagrangian-Eulerian scheme to handle the moving interface, demonstrate a strong effect of the elevated van der Waals attraction on precipitating tear-film breakup. The change in boundary condition on the cornea has a relatively minor role. Using realistic parameters, our heterogeneous mucin model is able to predict quantitatively the shortening of tear-film breakup time observed in diseased eyes.

Simulation of nanoparticle transport and adsorption in a microfluidic lung-on-a-chip device.

The effect of air-borne nanoparticles (NPs) on human health is an active area of research, with clinical relevance evidenced by the current COVID-19 pandemic. As in vitro models for such studies, lung-on-a-chip (LOAC) devices can represent key physical and physiological aspects of alveolar tissues. However, widespread adoption of the LOAC device for NP testing has been hampered by low intra-laboratory and inter-laboratory reproducibility. To complement ongoing experimental work, we carried out finite-element simulations of the deposition of NPs on the epithelial layer of a well-established LOAC design. We solved the Navier-Stokes equations for the fluid flow in a three-dimensional domain and studied the particle transport using Eulerian advection-diffusion for fine NPs and Lagrangian particle tracking for coarse NPs. Using Langmuir and Frumkin kinetics for surface adsorption and desorption, we investigated NP adsorption under different exercise and breath-holding patterns. Conditions mimicking physical exercise, through changes in air-flow volume and breathing frequency, enhance particle deposition. Puff profiles typical of smoking, with breath-holding between inhalation and exhalation, also increase particle deposition per breathing cycle. Lagrangian particle tracking shows Brownian motion and gravitational settling to be two key factors, which may cooperate or compete with each other for different particle sizes. Comparisons are made with experimental data where possible and they show qualitative and semi-quantitative agreement. These results suggest that computer simulations can potentially inform and accelerate the design and application of LOAC devices for analyzing particulate- and microbe-alveolar interactions.

A biomechanical model for the transendothelial migration of cancer cells.

We propose a biomechanical model for the extravasation of a tumor cell (TC) through the endothelium of a blood vessel. Based on prior in vitro observations, we assume that the TC extends a protrusion between adjacent endothelial cells (ECs) that adheres to the basement membrane via focal adhesions (FAs). As the protrusion grows in size and branches out, the actomyosin contraction along the stress fibers (SFs) inside the protrusion pulls the relatively rigid nucleus through the endothelial opening. We model the chemo-mechanics of the SFs and the FAs by following the kinetics of the active myosin motors and high-affinity integrins, subject to mechanical feedback. This is incorporated into a finite-element simulation of the extravasation process, with the contractile force pulling the nucleus of the TC against elastic resistance of the ECs. To account for the interaction between the TC nucleus and the endothelium, we consider two scenarios: solid-solid contact and lubrication by cytosol. The former gives a lower bound for the required contractile force to realize transmigration, while the latter provides a more realistic representation of the process. Using physiologically reasonable parameters, our model shows that the SF and FA ensemble can produce a contractile force on the order of 70 nN, which is sufficient to deform the ECs and enable transmigration. Furthermore, we use an atomic force microscope to measure the resistant force on a human bladder cancer cell that is pushed through an endothelium cultured in vitro. The magnitude of the required force turns out to be in the range of 70-100 nN, comparable to the model predictions.

A Rho-GTPase based model explains group advantage in collective chemotaxis of neural crest cells.

A cluster of neural crest cells (NCCs) may chemotax up a shallow external gradient to which a single cell is unresponsive. To explain this intriguing 'group advantage', we propose a chemo-mechanical model based on the signaling proteins Rac1 and RhoA. We represent each cell as a polygon with nodes connected by elastic membranes. Via reaction-diffusion on the membrane and exchange with their cytosolic pools, Rac1 and RhoA interact to produce cell polarization and repolarization subject to random noise. Mechanically, we represent cell motility via overdamped nodal motion subject to passive elastic membrane forces and active protrusive or contractile forces where Rac1 or RhoA dominates. The model reproduces the random walk of a single cell in a weak gradient and two modes of cell-cell interaction: contact inhibition of locomotion and co-attraction. The simultaneous action of contact inhibition and co-attraction suppresses random Rac1 bursts on the membrane and serves to preserve existing protrusions. This amounts to an emergent persistence of polarity that markedly enhances the ability of a cluster of NCCs to chemotax in a weak gradient against random noise, thereby giving rise to the group advantage.

Dynamics of PAR Proteins Explain the Oscillation and Ratcheting Mechanisms in Dorsal Closure.

We present a vertex-based model for Drosophila dorsal closure that predicts the mechanics of cell oscillation and contraction from the dynamics of the PAR proteins. Based on experimental observations of how aPKC, Par-6, and Bazooka translocate from the circumference of the apical surface to the medial domain, and how they interact with each other and ultimately regulate the apicomedial actomyosin, we formulate a system of differential equations that captures the key features of dorsal closure, including distinctive behaviors in its early, slow, and fast phases. The oscillation in cell area in the early phase of dorsal closure results from an intracellular negative feedback loop that involves myosin, an actomyosin regulator, aPKC, and Bazooka. In the slow phase, gradual sequestration of apicomedial aPKC by Bazooka clusters causes incomplete disassembly of the actomyosin network over each cycle of oscillation, thus producing a so-called ratchet. The fast phase of rapid cell and tissue contraction arises when medial myosin, no longer antagonized by aPKC, builds up in time and produces sustained contraction. Thus, a minimal set of rules governing the dynamics of the PAR proteins, extracted from experimental observations, can account for all major mechanical outcomes of dorsal closure, including the transitions between its three distinct phases.

Modeling cell intercalation during Drosophila germband extension.

Germband extension during Drosophila development is primarily driven by cell intercalation, which involves three key components: planar cell polarity, anisotropic myosin contractile forces on cellular junctions, and cellular deformation and movement. Prior experimental work probed each of these factors in depth, but the connection between them remains unclear. This paper presents an integrated chemomechanical model that combines the three factors into a coherent mathematical framework for studying cell intercalation in the germband tissue. The model produces the planar cell polarization of key proteins, including Rho-kinase, Bazooka and myosin, the development of anisotropic contractile forces, and subsequent cell deformation and rearrangement. Cell intercalation occurs through T1 transitions among four neighboring cells and rosettes involving six cells. Such six-cell rosettes entail stronger myosin-based contractile forces, and on average produce a moderately larger amount of germband extension than the T1 transitions. The resolution of T1 and rosettes is driven by contractile forces on junctions anterior and posterior to the assembly as well as the pulling force of the medial myosin in the anterior and posterior cells. The global stretching due to posterior midgut invagination also plays a minor role. These model predictions are in reasonable agreement with experimental observations.

A Rho-GTPase based model explains spontaneous collective migration of neural crest cell clusters.

We propose a model to explain the spontaneous collective migration of neural crest cells in the absence of an external gradient of chemoattractants. The model is based on the dynamical interaction between Rac1 and RhoA that is known to regulate the polarization, contact inhibition and co-attraction of neural crest cells. Coupling the reaction-diffusion equations for active and inactive Rac1 and RhoA on the cell membrane with a mechanical model for the overdamped motion of membrane vertices, we show that co-attraction and contact inhibition cooperate to produce persistence of polarity in a cluster of neural crest cells by suppressing the random onset of Rac1 hotspots that may mature into new protrusion fronts. This produces persistent directional migration of cell clusters in corridors. Our model confirms a prior hypothesis that co-attraction and contact inhibition are key to spontaneous collective migration, and provides an explanation of their cooperative working mechanism in terms of Rho GTPase signaling. The model shows that the spontaneous migration is more robust for larger clusters, and is most efficient in a corridor of optimal confinement.

Asymmetric drop coalescence launches fungal ballistospores with directionality.

Thousands of fungal species use surface energy to power the launch of their ballistospores. The surface energy is released when a spherical Buller's drop at the spore's hilar appendix merges with a flattened drop on the adaxial side of the spore. The launching mechanism is primarily understood in terms of energetic models, and crucial features such as launching directionality are unexplained. Integrating experiments and simulations, we advance a mechanistic model based on the capillary-inertial coalescence between the Buller's drop and the adaxial drop, a pair that is asymmetric in size, shape and relative position. The asymmetric coalescence is surprisingly effective and robust, producing a launching momentum governed by the Buller's drop and a launching direction along the adaxial plane of the spore. These key functions of momentum generation and directional control are elucidated by numerical simulations, demonstrated on spore-mimicking particles, and corroborated by published ballistospore kinematics. Our work places the morphological and kinematic diversity of ballistospores into a general mechanical framework, and points to a generic catapulting mechanism of colloidal particles with implications for both biology and engineering.

Capillary-inertial colloidal catapults upon drop coalescence.

Surface energy released upon drop coalescence is known to power the self-propelled jumping of liquid droplets on superhydrophobic solid surfaces, and the jumping droplets can additionally carry colloidal payloads toward self-cleaning. Here, we show that drop coalescence on a spherical particle leads to self-propelled launching of the particle from virtually any solid surface. The main prerequisite is an intermediate wettability of the particle, such that the momentum from the capillary-inertial drop coalescence process can be transferred to the particle. By momentum conservation, the launching velocity of the particle-drop complex is proportional to the capillary-inertial velocity based on the drop radius and to the fraction of the liquid mass in the total mass. The capillary-inertial catapult is not only an alternative mechanism for removing colloidal contaminants, but also a useful model system for studying ballistospore launching.

Modeling the Mechanosensitivity of Neutrophils Passing through a Narrow Channel.

Recent experiments have found that neutrophils may be activated after passing through microfluidic channels and filters. Mechanical deformation causes disassembly of the cytoskeleton and a sudden drop of the elastic modulus of the neutrophil. This fluidization is followed by either activation of the neutrophil with protrusion of pseudopods or a uniform recovery of the cytoskeleton network with no pseudopod. The former occurs if the neutrophil traverses the narrow channel at a slower rate. We propose a chemo-mechanical model for the fluidization and activation processes. Fluidization is treated as mechanical destruction of the cytoskeleton by sufficiently rapid bending. Loss of the cytoskeleton removes a pathway by which cortical tension inhibits the Rac protein. As a result, Rac rises and polarizes through a wave-pinning mechanism if the chemical reaction rate is fast enough. This leads to recovery and reinforcement of the cytoskeleton at the front of the neutrophil, and hence protrusion and activation. Otherwise the Rac signal returns to a uniform pre-deformation state and no activation occurs. Thus, mechanically induced neutrophil activation is understood as the competition between two timescales: that of chemical reaction and that of mechanical deformation. The model captures the main features of the experimental observation.

A biomechanical model for cell polarization and intercalation during Drosophila germband extension.

Germband extension during Drosophila development features the merging of cells along the dorsal-ventral (DV) axis and their separation along the anterior-posterior (AP) axis. This intercalation process involves planar cell polarity, anisotropic contractile forces along cell edges, and concerted cell deformation and movement. Although prior experiments have probed each of these factors separately, the connection among them remains unclear. This paper presents a chemo-mechanical model that integrates the three factors into a coherent framework. The model predicts the polarization of Rho-kinase, myosin and Bazooka downstream of an anisotropic Shroom distribution. In particular, myosin accumulates on cell edges along the DV axis, causing them to contract into a vertex. Subsequently, medial myosin in the cells anterior and posterior to the vertex helps to elongate it into a new edge parallel to the body axis. Thus, the tissue extends along the AP axis and narrows in the transverse direction through neighbor exchange. Model predictions of the polarity of the proteins and cell and tissue deformation are in good agreement with experimental observations.