Measuring the Transmembrane Registration of Lipid Domains in Droplet Interface Bilayers through Tensiometry.

Diverse collections of lipids self-assemble into domains within biological membranes, and these domains are typically organized in both the transverse and lateral directions of the membrane. The ability of the membrane to link these domains across the membrane's interior grants cells control over features on the external cellular surface. Numerous hypothesized factors drive the cross-membrane (or transverse) coupling of lipid domains. In this work we seek to isolate these transverse lipid-lipid influences in a simple model system using droplet interface bilayers (DIBs) to better understand the associated mechanics. DIBs enable symmetric and asymmetric combinations of domain-forming lipid mixtures within a model bilayer, and the evolving energetics of the membrane may be tracked using drop-shape analysis. We find that symmetric distributions of domain-forming lipids produce long-lasting, gradual shifts in the DIB membrane energetics that are not observed in asymmetric distributions of the lipids where the domain-forming lipids are only within one leaflet. The approach selected for this work provides experimental measurement of the mismatch penalty associated with antiregistered lipid domains as well as measurements of the influence of rafts on DIB behaviors with suggestions for their future use as a model platform.

Enhancing membrane-based soft materials with magnetic reconfiguration events.

Adaptive and bioinspired droplet-based materials are built using the droplet interface bilayer (DIB) technique, assembling networks of lipid membranes through adhered microdroplets. The properties of these lipid membranes are linked to the properties of the droplets forming the interface. Consequently, rearranging the relative positions of the droplets within the network will also alter the properties of the lipid membranes formed between them, modifying the transmembrane exchanges between neighboring compartments. In this work, we achieved this through the use of magnetic fluids or ferrofluids selectively dispersed within the droplet-phase of DIB structures. First, the ferrofluid DIB properties are optimized for reconfiguration using a coupled experimental-computational approach, exploring the ideal parameters for droplet manipulation through magnetic fields. Next, these findings are applied towards larger, magnetically-heterogeneous collections of DIBs to investigate magnetically-driven reconfiguration events. Activating electromagnets bordering the DIB networks generates rearrangement events by separating and reforming the interfacial membranes bordering the dispersed magnetic compartments. These findings enable the production of dynamic droplet networks capable of modifying their underlying membranous architecture through magnetic forces.

Studying the Mechanics of Membrane Permeabilization through Mechanoelectricity.

In this research, real-time monitoring of lipid membrane disruption is made possible by exploiting the dynamic properties of model lipid bilayers formed at oil-water interfaces. This involves tracking an electrical signal generated through rhythmic membrane perturbation translated into the adsorption and penetration of charged species within the membrane. Importantly, this allows for the detection of membrane surface interactions that occur prior to pore formation that may be otherwise undetected. The requisite dynamic membranes for this approach are made possible through the droplet interface bilayer (DIB) technique. Membranes are formed at the interface of lipid monolayer-coated aqueous droplets submerged in oil. We present how cyclically alternating the membrane area leads to the generation of mechanoelectric current. This current is negligible without a transmembrane voltage until a composition mismatch between the membrane monolayers is produced, such as a one-sided accumulation of disruptive agents. The generated mechanoelectric current is then eliminated when an applied electric field compensates for this asymmetry, enabling measurement of the transmembrane potential offset. Tracking the compensating voltage with respect to time then reveals the gradual accumulation of disruptive agents prior to membrane permeabilization. The innovation of this work is emphasized in its ability to continuously track membrane surface activity, highlighting the initial interaction steps of membrane disruption. In this paper, we begin by validating our proposed approach against measurements taken for fixed composition membranes using standard electrophysiological techniques. Next, we investigate surfactant adsorption, including hexadecyltrimethylammonium bromide (CTAB, cationic) and sodium decyl sulfate (SDS, anionic), demonstrating the ability to track adsorption prior to disruption. Finally, we investigate the penetration of lipid membranes by melittin, confirming that the peptide insertion and disruption mechanics are, in part, modulated by membrane composition.

A skin-inspired soft material with directional mechanosensation.

Lessons about artificial sensor design may be taken from evolutionarily perfected physiological systems. Mechanosensory cells in human skin are exquisitely sensitive to gentle touch and enable us to distinguish objects of different stiffnesses and textures. These cells are embedded in soft epidermal layers of gel-like consistency. Reproducing these mechanosensing capabilities in new soft materials may lead to the development of adaptive mechanosensors which will further enhance the abilities of engineered membrane-based structures with bioinspired sensing strategies. This strategy is explored here using droplet interface bilayers embedded within a thermoreversible organogel. The interface between two lipid-coated aqueous inclusions contained within a soft polymeric matrix forms a lipid bilayer resembling the lipid matrix of cell membranes. These interfaces are functionalized with bacterial mechanosensitive channels (V23T MscL) which convert membrane tension into changes in membrane conductance, mimicking mechanosensitive channel activation in mammalian mechanosensory cells. The distortion of encapsulated adhered droplets by cyclical external forces are first explored using a finite element composite model illustrating the directional propagation of mechanical disturbances imposed by a piston. The model predicts that the orientation of the droplet pair forming the membrane relative to the direction of the compression plays a role in the membrane response. The directional dependence of mechanosensitive channel activation in response to gel compression is confirmed experimentally and shows that purely compressive perturbations normal to the interface invoke different channel activities as compared to shearing displacement along a plane of the membrane. The developed system containing specially positioned pairs of droplets functionalized with bacterial mechanosensitive channels and embedded in a gel creates a skin-inspired soft material with a directional response to mechanical perturbation.

Droplet-Based Membranous Soft Materials.

Inspired by the structure and functionality of natural cellular tissues, droplet interface bilayer (DIB)-based materials strategically combine model membrane assembly techniques and droplet microfluidics. These structures have shown promising results in applications ranging from biological computing to chemical microrobots. This Feature Article briefly explores recent advances in the areas of construction, manipulation, and functionalization of DIB networks; discusses their unique mechanics; and focuses on the contributions of our lab in the advancement of this platform. We also reflect on some of the limitations facing DIB-based materials and how they might be addressed, highlighting promising applications made possible through the refinement of the material concept.

Evaluating the therapeutic potential of ADAR1 inhibition for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of endogenous double-stranded RNAs. ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that expression of ADAR1, specifically its p150 isoform, is required for the survival of TNBC cell lines. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. Moreover, ADAR1 knockdown leads to robust translational repression. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescued the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.

A new approach for investigating the response of lipid membranes to electrocompression by coupling droplet mechanics and membrane biophysics.

A new method for quantifying lipid-lipid interactions within biomimetic membranes undergoing electrocompression is demonstrated by coupling droplet mechanics and membrane biophysics. The membrane properties are varied by altering the lipid packing through the introduction of cholesterol. Pendant drop tensiometry is used to measure the lipid monolayer tension at an oil-water interface. Next, two lipid-coated aqueous droplets are manipulated into contact to form a bilayer membrane at their adhered interface. The droplet geometries are captured from two angles to provide accurate measurements of both the membrane area and the contact angle between the adhered droplets. Combining the monolayer tension and contact angle measurements enables estimations of the membrane tension with respect to lipid composition. Then, the membrane is electromechanically compressed using a transmembrane voltage. Electrostatic pressure, membrane tension and the work necessary for bilayer thinning are tracked, and a model is proposed to capture the mechanics of membrane compression. The results highlight that a previously unaccounted for energetic term is produced during compression, potentially reflecting changes in the lateral membrane structure. This residual energy is eliminated in cases with cholesterol mole fractions of 0.2 and higher, suggesting that cholesterol diminishes these adjustments.

Photopolymerized microdomains in both lipid leaflets establish diffusive transport pathways across biomimetic membranes.

Controlled transport within a network of aqueous subcompartments provides a foundation for the construction of biologically-inspired materials. These materials are commonly assembled using the droplet interface bilayer (DIB) technique, adhering droplets together into a network of lipid membranes. DIB structures may be functionalized to generate conductive pathways by enhancing the permeability of pre-selected membranes, a strategy inspired by nature. Traditionally these pathways are generated by dissolving pore-forming toxins (PFTs) in the aqueous phase. A downside of this approach when working with larger DIB networks is that transport is enabled in all membranes bordering the droplets containing the PFT, instead of occurring exclusively between selected droplets. To rectify this limitation, photopolymerizable phospholipids (23:2 DiynePC) are incorporated within the aqueous phase of the DIB platform, forming conductive pathways in the lipid membranes post-exposure to UV-C light. Notably these pathways are only formed in the membrane if both adhered droplets contain the photo-responsive lipids. Patterned DIB networks can then be generated by controlling the lipid composition within select droplets which creates conductive routes one droplet thick. We propose that the incorporation of photo-polymerizable phospholipids within the aqueous phase of DIB networks will improve the resolution of the patterned conductive pathways and reduce diffusive loss within the synthetic biological network.

Hydrogel Microelectrodes for the Rapid, Reliable, and Repeatable Characterization of Lipid Membranes.

Model lipid bilayer membranes provide approximations of natural cellular membranes that may be formed in the laboratory to study their mechanics and interactions with the surrounding environment. A new approach for their formation is proposed here based on the self-assembly of lipid monolayers at oil-water interfaces, creating a lipid-coated hydrogel-tipped electrode that produces a stable lipid membrane on the surface when introduced to a lipid-coated aqueous droplet. Membrane formation using the hydrogel microelectrode is tested for a variety of lipids and oils. The channel-forming peptide alamethicin is added to the membrane, and its functionality is verified. Finally, asymmetric membranes are created using varying lipid compositions, and the capacity for repeated quantification of membrane structure is demonstrated. The proposed hydrogel microelectrodes are compatible with multiple oils and lipids, simple to use, and suitable for detecting the presence of both biomolecular transporters and dissolved lipid compositions within aqueous droplets.

Evaluation of bending modulus of lipid bilayers using undulation and orientation analysis.

In the current paper, phospholipid bilayers are modeled using coarse-grained molecular dynamics simulations with the MARTINI force field. The extracted molecular trajectories are analyzed using Fourier analysis of the undulations and orientation vectors to establish the differences between the two approaches for evaluating the bending modulus. The current work evaluates and extends the implementation of the Fourier analysis for molecular trajectories using a weighted horizon-based averaging approach. The effect of numerical parameters in the analysis of these trajectories is explored by conducting parametric studies. Computational modeling results are validated against experimentally characterized bending modulus of lipid membranes using a shape fluctuation analysis. The computational framework is then used to estimate the bending moduli for different types of lipids (phosphocholine, phosphoethanolamine, and phosphoglycerol). This work provides greater insight into the numerical aspects of evaluating the bilayer bending modulus, provides validation for the orientation analysis technique, and explores differences in bending moduli based on differences in the lipid nanostructures.

Encapsulating Networks of Droplet Interface Bilayers in a Thermoreversible Organogel.

The development of membrane-based materials that exhibit the range and robustness of autonomic functions found in biological systems remains elusive. Droplet interface bilayers (DIBs) have been proposed as building blocks for such materials, owing to their simplicity, geometry, and capability for replicating cellular phenomena. Similar to how individual cells operate together to perform complex tasks and functions in tissues, networks of functionalized DIBs have been assembled in modular/scalable networks. Here we present the printing of different configurations of picoliter aqueous droplets in a bath of thermoreversible organogel consisting of hexadecane and SEBS triblock copolymers. The droplets are connected by means of lipid bilayers, creating a network of aqueous subcompartments capable of communicating and hosting various types of chemicals and biomolecules. Upon cooling, the encapsulating organogel solidifies to form self-supported liquid-in-gel, tissue-like materials that are robust and durable. To test the biomolecular networks, we functionalized the network with alamethicin peptides and alpha-hemolysin (αHL) channels. Both channels responded to external voltage inputs, indicating the assembly process does not damage the biomolecules. Moreover, we show that the membrane properties may be regulated through the deformation of the surrounding gel.

Reconfiguring droplet interface bilayer networks through sacrificial membranes.

The droplet interface bilayer platform allows for the fabrication of stimuli-responsive microfluidic materials, using phospholipids as an organic surfactant in water-in-oil mixtures. In this approach, lipid-coated droplets are adhered together in arranged networks, forming lipid bilayer membranes with embedded transporters and establishing selective exchange pathways between neighboring aqueous subcompartments. The resulting material is a biologically inspired droplet-based material that exhibits emergent properties wherein different droplets accomplish different functions, similar to multicellular organisms. These networks have been successfully applied towards biomolecular sensing and energy harvesting applications. However, unlike their source of inspiration, these droplet structures are often static. This limitation not only renders the networks unable to adapt or modify their structure and function after formation but also limits their long term use as passive ionic exchange between neighboring droplet pairs may initiate immediately after the membranes are established. This work addresses this shortcoming by rupturing selected sacrificial membranes within the collections of droplets to rearrange the remaining droplets into new configurations, redirecting the droplet-droplet exchange pathways. This is accomplished through electrical shocks applied between selected droplets. Experimental outcomes are compared to predictions provided by a coupled mechanical-electrical model for the droplet networks, and then advanced configurations are proposed using this model.

Ferrofluid-Based Droplet Interface Bilayer Networks.

Droplet interface bilayer (DIB) networks allow for the construction of stimuli-responsive, membrane-based materials. Traditionally used for studying cellular transport phenomena, the DIB technique has proven its practicality when creating structured droplet networks. These structures consist of aqueous compartments capable of exchanging their contents across membranous barriers in a regulated fashion via embedded biomolecules, thus approximating the activity of natural cellular systems. However, lipid bilayer networks are often static and incapable of any reconfiguration in their architecture. In this study, we investigate the incorporation of a magnetic fluid or ferrofluid within the droplet phases for the creation of magnetically responsive DIB arrays. The impact of adding ferrofluid to the aqueous phases of the DIB networks is assessed by examining the bilayers' interfacial tensions, thickness, and channel activity. Once compatibility is established, potential applications of the ferrofluid-enabled DIBs are showcased by remotely modifying membrane qualities through magnetic fields. Ferrofluids do not significantly alter the bilayers' properties or functionality and can therefore be safely embedded within the DIB platform, allowing for remote manipulation of the interfacial bilayer properties.

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein.

Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a ß-fibrillizing peptide (ßFP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a ßFP that forms ß-sheet fibrils at high salt concentrations. The IgG-binding, fluorogenic film can be injected in vivo through conventional needled syringes. Confocal microscopic images and dose-response titration experiments showed that loading of IgG into the film was mediated by pAG(MG) bound to the FAP. Release of IgG in vitro was significantly delayed by the bioaffinity mechanism; 26% of the IgG were released from films embedded with pAG(MG) after five days, compared to close to 90% in films without pAG(MG). Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAG(MG). When injected into the subcutaneous space of mouse footpads, film-embedded IgG were retained locally, with distribution through the lymphatics impeded. The ability to track IgG binding sites and distribution simultaneously will aid the optimization of local antibody delivery systems.

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers.

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Multiscale modeling of droplet interface bilayer membrane networks.

Droplet interface bilayer (DIB) networks are considered for the development of stimuli-responsive membrane-based materials inspired by cellular mechanics. These DIB networks are often modeled as combinations of electrical circuit analogues, creating complex networks of capacitors and resistors that mimic the biomolecular structures. These empirical models are capable of replicating data from electrophysiology experiments, but these models do not accurately capture the underlying physical phenomena and consequently do not allow for simulations of material functionalities beyond the voltage-clamp or current-clamp conditions. The work presented here provides a more robust description of DIB network behavior through the development of a hierarchical multiscale model, recognizing that the macroscopic network properties are functions of their underlying molecular structure. The result of this research is a modeling methodology based on controlled exchanges across the interfaces of neighboring droplets. This methodology is validated against experimental data, and an extension case is provided to demonstrate possible future applications of droplet interface bilayer networks.

Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.

MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

Modeling the proton sponge hypothesis: examining proton sponge effectiveness for enhancing intracellular gene delivery through multiscale modeling.

Dendrimers have been proposed as therapeutic gene delivery platforms. Their superior transfection efficiency is attributed to their ability to buffer the acidification of the endosome and attach to the nucleic acids. For effective transfection, the strategy is to synthesize novel dendrimers that optimize both of these traits, but the prediction of the buffering behavior in the endosome remains elusive. It is suggested that buffering dendrimers induce an osmotic pressure sufficient to rupture the endosome and release nucleic acids, which forms to sequestrate most internalized exogenous materials. Presented here are the results of a computational study modeling osmotically driven endosome burst or the 'proton sponge effect.' The approach builds on previous cellular simulation efforts by linking the previous model with a sponge protonation model, then observing the impact on endosomal swelling and acidification. Calibrated and validated using reported experimental data, the simulations offer insights into defining the properties of suitable dendrimers for enhancing gene delivery as a function of polymer structure.