The Paradoxical Behavior of Rough Colloids at Fluid Interfaces.

Colloidal particles adsorb and remain trapped at immiscible fluid interfaces due to strong interfacial adsorption energy with a contact angle defined by the chemistry of the particle and fluid phases. An undulated contact line may appear due to either particle surface roughness or shape anisotropy, which results in a quadrupolar interfacial deformation and strong long-range capillary interaction between neighboring particles. While each effect has been observed separately, here we report the paradoxical impact of surface roughness on spherical and anisotropic ellipsoidal polymer colloids. Using a seeded emulsion polymerization technique, we synthesize spherical and ellipsoidal particles with controlled roughness magnitudes and topography (convex/concave). Via in situ measurement of the interfacial deformation around colloids at an air-water interface, we find that while surface roughness strengthens the quadrupolar deformation in spheres as expected by theory, in stark contrast, it weakens the same in ellipsoids. As roughness increases, particles of both shapes become more hydrophilic, and their apparent contact angle decreases. Using numerical predictions, we show that this partially explains the decreased interfacial deformation and capillary interactions between the ellipsoids. Therefore, particle surface engineering has the potential to decrease the capillary deformation by asymmetric particles via changing their capillary pinning, as well as wetting behavior at fluid interfaces.

Effects of Ion Concentration and Headgroup Chemistry on Thin Lipid Film Drainage.

While the use of lipid nanoparticles in drug delivery applications has grown over the past few decades, much work remains to be done toward the characterization and rational design of the drug carriers. A key feature of delivery is the interaction of the exterior leaflet of the LNP with the outer leaflet of the cell membrane, which relies in part on the fusogenicity of the lipids and the ionic environment. In this paper, we study the interactions between two lipid monolayers using a thin film balance to create lipid thin films and interferometry to measure film evolution. We probe the role of lipid headgroup chemistry and charge, along with ionic solution conditions, in either promoting or hindering film drainage and stability. Specific headgroups phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylserine (PS) are chosen to represent a combination of charge and fusogenicity. We quantify each film's drainage characteristics over a range of capillary numbers. Qualitatively, we find that films transition from drainage via a large dimple to drainage via channels and vortices as the capillary number increases. Additionally, we observe a transition from electrostatically dominated film drainage at low CaCl2 concentrations to fusogenic-dominated film drainage at higher CaCl2 concentrations for anionic fusogenic (PS) films. Understanding the role of headgroup composition, ionic composition, and ionic concentration will pave the way for the design of tunable vesicle and buffer systems that behave desirably across a range of ex vivo and in vivo environments.

Effects of crowding on the diffusivity of membrane adhered particles.

The lateral diffusion of cell membrane inclusions, such as integral membrane proteins and bound receptors, drives critical biological processes, including the formation of complexes, cell-cell signaling, and membrane trafficking. These diffusive processes are complicated by how concentrated, or "crowded", the inclusions are, which can occupy between 30-50% of the area fraction of the membrane. In this work, we elucidate the effects of increasing concentration of model membrane inclusions in a free-standing artificial cell membrane on inclusion diffusivity and the apparent viscosity of the membrane. By multiple particle tracking of fluorescent microparticles covalently tethered to the bilayer, we show the transition from expected Brownian dynamics, which accurately measure the membrane viscosity, to subdiffusive behavior with decreased diffusion coefficient as the particle area fraction increases from 1% to around 30%, approaching physiological levels of crowding. At high crowding, the onset of non-Gaussian behavior is observed. Using hydrodynamic models relating the 2D diffusion coefficient to the viscosity of a membrane, we determine the apparent viscosity of the bilayer from the particle diffusivity and show an increase in the apparent membrane viscosity with increasing particle area fraction. However, the scaling of this increase is in contrast with the behavior of monolayer inclusion diffusion and bulk suspension rheology. These results demonstrate that physiological levels of model membrane crowding nontrivially alter the dynamics and apparent viscosity of the system, which has implications for understanding membrane protein interactions and particle-membrane transport processes.

Engineering the surface patchiness and topography of polystyrene colloids: From spheres to ellipsoids.

HYPOTHESIS: Colloidal surface morphology determines suspension properties and applications. While existing methods are effective at generating specific features on spherical particles, an approach extending this to non-spherical particles is currently missing. Synthesizing un-crosslinked polymer microspheres with controlled chemical patchiness would allow subsequent thermomechanical stretching to translate surface topographical features to ellipsoidal particles. EXPERIMENTS: A systematic study using seeded emulsion polymerization to create polystyrene (PS) microspheres with controlled surface patches of poly(tert-butyl acrylate) (PtBA) was performed with different polymerization parameters such as concentration of tBA monomer, co-swelling agent, and initiator. Thermomechanical stretching converted seed spheres to microellipsoids. Acid catalyzed hydrolysis (ACH) was performed to remove the patch domains. Roughness was characterized before and after ACH using atomic force microscopy. FINDINGS: PS spheres with controlled chemical patchiness were synthesized. A balance between two factors, domain coalescence from reduced viscosity and domain growth via monomer absorption, dictates the final PtBA) patch features. ACH mediated removal of patch domains produced either golf ball-like porous particles or multicavity particles, depending on the size of the precursor patches. Patchy microspheres were successfully stretched into microellipsoids while retaining their surface characteristics. Particle roughness is governed by the patch geometry and increases after ACH. Overall, this study provides a facile yet controllable platform for creating colloids with highly adjustable surface patterns.

Nanoscale Porosity in Microellipsoids Cloaks Interparticle Capillary Attraction at Fluid Interfaces.

Anisotropic particles pinned at fluid interfaces tend toward disordered multiparticle configurations due to large, orientationally dependent, capillary forces, which is a significant barrier to exploiting these particles to create functional self-assembled materials. Therefore, current interfacial assembly methods typically focus on isotropic spheres, which have minimal capillary attraction and no dependence on orientation in the plane of the interface. In order to create long-range ordered structures with complex configurations via interfacially trapped anisotropic particles, control over the interparticle interaction energy via external fields and/or particle engineering is necessary. Here, we synthesize colloidal ellipsoids with nanoscale porosity and show that their interparticle capillary attraction at a water-air interface is reduced by an order of magnitude compared to their smooth counterparts. This is accomplished by comparing the behavior of smooth, rough, and porous ellipsoids at a water-air interface. By monitoring the dynamics of two particles approaching one another, we show that the porous particles exhibit a much shorter-range capillary interaction potential, with scaling intriguingly different than theory describing the behavior of smooth ellipsoids. Further, interferometry measurements of the fluid deformation surrounding a single particle shows that the interface around porous ellipsoids does not possess the characteristic quadrupolar symmetry of smooth ellipsoids, and quantitatively confirms the decrease in capillary interaction energy. By engineering nanostructured surface features in this fashion, the interfacial capillary interactions between particles may be controlled, informing an approach for the self-assembly of complex two-dimensional microstructures composed of anisotropic particles.

Determining the Bending Rigidity of Free-Standing Planar Phospholipid Bilayers.

We describe a method to determine membrane bending rigidity from capacitance measurements on large area, free-standing, planar, biomembranes. The bending rigidity of lipid membranes is an important biological mechanical property that is commonly optically measured in vesicles, but difficult to quantify in a planar, unsupported system. To accomplish this, we simultaneously image and apply an electric potential to free-standing, millimeter area, planar lipid bilayers composed of DOPC and DOPG phospholipids to measure the membrane Young's (elasticity) modulus. The bilayer is then modeled as two adjacent thin elastic films to calculate bending rigidity from the electromechanical response of the membrane to the applied field. Using DOPC, we show that bending rigidities determined by this approach are in good agreement with the existing work using neutron spin echo on vesicles, atomic force spectroscopy on supported lipid bilayers, and micropipette aspiration of giant unilamellar vesicles. We study the effect of asymmetric calcium concentration on symmetric DOPC and DOPG membranes and quantify the resulting changes in bending rigidity. This platform offers the ability to create planar bilayers of controlled lipid composition and aqueous ionic environment, with the ability to asymmetrically alter both. We aim to leverage this high degree of compositional and environmental control, along with the capacity to measure physical properties, in the study of various biological processes in the future.

Domain Size Regulation in Phospholipid Model Membranes Using Oil Molecules and Hybrid Lipids.

The formation of domains in multicomponent lipid mixtures has been suggested to play a role in moderating signal transduction in cells. Understanding how domain size may be regulated by both hybrid lipid molecules and impurities is important for understanding real biological processes; at the same time, developing model systems where domain size can be regulated is crucial to enable systematic studies of domain formation kinetics and thermodynamics. Here, we perform a model study of the effects of oil molecules, which swell the bilayer, and line-active hybrid phospholipids using a thermally induced liquid-solid phase separation in planar, free-standing lipid bilayers consisting of DOPC and DPPC (1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, respectively). The experiments show that the kinetics of domain growth are significantly affected by the type and molecular structure of the oil (squalene, hexadecane, or decane), with the main contributing factors being the degree of swelling of the bilayer and the changes in line tension induced by the different oils, with smaller domains resulting from systems with smaller values of the line tension. POPC (1-palmitoyl-sn-2-oleoyl-glycero-3-phosphocholine), on the other hand, acts as a line-active hybrid lipid, reducing the domain size when added in small amounts and slowing down domain coarsening. Finally, we show that despite the regulation of domain size by both methods, the phase transition temperature is influenced by the presence of oil molecules but not significantly by the presence of hybrid lipids. Overall, our results show how to regulate domain size in binary membrane model systems, over a wide range of length scales, by incorporating oil molecules and hybrid lipids.

Fabrication and electromechanical characterization of freestanding asymmetric membranes.

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DOPG) content as compared with previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC-DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison with symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.

Gradient stretching to produce variable aspect ratio colloidal ellipsoids.

Developing reliable synthetic methods for producing shape-anisotropic polymer colloids is essential for their use in novel functional materials. In designing such materials from ellipsoidal particles, it is often necessary to screen a wide range of particle sizes and aspect ratios to appropriately understand how microscopic particle characteristics dictate macroscopic material response. Here, we describe a technique to simultaneously produce a broad range of aspect ratio polymer ellipsoid samples from a single synthetic step. The technique extends the traditional film-stretching approach to create ellipsoids by introducing a gradient in strain and film cooling, which results in varying degrees of particle stretching. We empirically calibrate the device such that the final particle elongation may be predicted from the film characteristics, enabling the selective harvesting of ellipsoids with desired dimensions and which can be isolated by aspect ratio. The method is applied successfully to a wide range of seed particle diameters (500 nm - 10 µm) and enables the rapid synthesis of variable aspect ratio particles for systematic studies of anisotropic particles.

Toward Realistic Large-Area Cell Membrane Mimics: Excluding Oil, Controlling Composition, and Including Ion Channels.

Capacitance measurements provide unique insights into the thickness, compressibility, and composition of large-area membrane bilayers and are used here in addition to demonstrate the successful incorporation of model ion channels. The simultaneous ability to control the bilayer size, manipulate tension, and optically monitor and electrically stimulate freestanding membranes enables precise determination of their specific capacitance and thickness across a wide range of areas. We confirm that membranes formed by this recently developed technique have capacitive properties similar to those formed by existing protocols, including solvent-free approaches, and discuss the effect using either hexadecane or squalene as the oil solvent. The results obtained here are relevant for other methods where lipid membranes are reconstituted from a bulk oil solvent. Because biological membranes have a diverse phospholipid profile, we show that the technique can successfully reconstitute membranes with binary composition mixtures. As an outlook, we show the capability of model membrane proteins, specifically α-hemolysin and alamethicin, to be incorporated into the formed bilayers and measure ion transport.

Arresting dissolution by interfacial rheology design.

A strategy to halt dissolution of particle-coated air bubbles in water based on interfacial rheology design is presented. Whereas previously a dense monolayer was believed to be required for such an "armored bubble" to resist dissolution, in fact engineering a 2D yield stress interface suffices to achieve such performance at submonolayer particle coverages. We use a suite of interfacial rheology techniques to characterize spherical and ellipsoidal particles at an air-water interface as a function of surface coverage. Bubbles with varying particle coverages are made and their resistance to dissolution evaluated using a microfluidic technique. Whereas a bare bubble only has a single pressure at which a given radius is stable, we find a range of pressures over which bubble dissolution is arrested for armored bubbles. The link between interfacial rheology and macroscopic dissolution of [Formula: see text] 100 [Formula: see text]m bubbles coated with [Formula: see text] 1 [Formula: see text]m particles is presented and discussed. The generic design rationale is confirmed by using nonspherical particles, which develop significant yield stress at even lower surface coverages. Hence, it can be applied to successfully inhibit Ostwald ripening in a multitude of foam and emulsion applications.

Molecular, Local, and Network-Level Basis for the Enhanced Stiffness of Hydrogel Networks Formed from Coassembled Racemic Peptides: Predictions from Pauling and Corey.

Hydrogels prepared from self-assembling peptides are promising materials for medical applications, and using both l- and d-peptide isomers in a gel's formulation provides an intuitive way to control the proteolytic degradation of an implanted material. In the course of developing gels for delivery applications, we discovered that a racemic mixture of the mirror-image ß-hairpin peptides, named MAX1 and DMAX1, provides a fibrillar hydrogel that is four times more rigid than gels formed by either peptide alone-a puzzling observation. Herein, we use transmission electron microscopy, small angle neutron scattering, solid state NMR, diffusing wave, infrared, and fluorescence spectroscopies, and modeling to determine the molecular basis for the increased mechanical rigidity of the racemic gel. We find that enantiomeric peptides coassemble in an alternating fashion along the fibril long axis, forming an extended heterochiral pleat-like ß-sheet, a structure predicted by Pauling and Corey in 1953. Hydrogen bonding between enantiomers within the sheet dictates the placement of hydrophobic valine side chains in the fibrils' dry interior in a manner that allows the formation of nested hydrophobic interactions between enantiomers, interactions not accessible within enantiomerically pure fibrils. Importantly, this unique molecular arrangement of valine side chains maximizes inter-residue contacts within the core of the fibrils resulting in their local stiffening, which in turn, gives rise to the significant increase in bulk mechanical rigidity observed for the racemic hydrogel.

Colloidal Switches by Electric and Magnetic Fields.

External electric and magnetic fields have already been proven to be a versatile tool to control the particle assembly; however, the degree of control of the dynamics and versatility of the produced structures is expected to increase if both can be implemented simultaneously. For example, while micromagnets can rapidly assemble superparamagnetic particles, repeated, rapid disassembly or reassembly is not trivial because of the remanence and coercivity of metals used in such applications. Here, an interdigitated design of micromagnet and microfabricated electrodes enables rapid switching of colloids between their magnetic and electric potential minima. Active control over colloids between two such adjacent potential minima enables a fast on/off mechanism, which is potentially important for optical switches or display technologies. Moreover, we demonstrate that the response time of the colloids between these states is on the order of tens of milliseconds, which is tunable by electric field strength. By carefully designing the electrode pattern, our strategy enables the switchable assembly of single particles down to few microns and also hierarchical assemblies containing many particles. Our work on precise dynamic control over the particle position would open new avenues to find potential applications in optical switches and display technologies.

Millimeter-area, free standing, phospholipid bilayers.

Minimal model biomembrane studies have the potential to unlock the fundamental mechanisms of cellular function that govern the processes upon which life relies. However, existing methods to fabricate free-standing model membranes currently have significant limitations. Bilayer sizes are often tens of micrometers, decoupling curvature or substrate effects, orthogonal control over tension, and solvent exchange combined with microscopy techniques is not possible, which restricts the studies that can be performed. Here, we describe a versatile platform to generate free standing, planar, phospholipid bilayers with millimeter scale areas. The technique relies on an adapted thin-film balance apparatus allowing for the dynamic control of the nucleation and growth of a planar black lipid membrane in the center of an orifice surrounded by microfluidic channels. Success is demonstrated using several different lipid types, including mixtures that show the same temperature dependent phase separation as existing protocols, moreover, membranes are highly stable. Two advantages unique to the proposed method are the dynamic control of the membrane tension and the possibility to make extremely large area membranes. We demonstrate this by showing how a block polymer, F68, used in drug delivery increases the membrane compliance. Together, the results demonstrate a new paradigm for studying the mechanics, structure, and function of model membranes.

Simple Optical Imaging of Nanoscale Features in Free-Standing Films.

Measuring thicknesses in thin films with high spatial and temporal resolution is of prime importance for understanding the structure and dynamics in thin films and membranes. In the present work, we introduce fluorescence-interferometry, a method that combines standard reflected light thin film interferometry with simultaneous fluorescence measurements. We apply this method to the thinning dynamics and phase separation in free-standing inverse phospholipid bilayer films. The measurements were carried out using a standard fluorescence microscope using multichannel imaging and yielded subnanometer resolution, which is applied to optically measure the discrete thickness variations across phase-separated membranes.

Anisotropic hypersonic phonon propagation in films of aligned ellipsoids.

A material with anisotropic elastic mechanical properties and a direction-dependent hypersonic band gap is fabricated using ac electric field-directed convective self-assembly of colloidal ellipsoids. The frequency of the gap, which is detected in the direction perpendicular to particle alignment and entirely absent parallel to alignment, and the effective sound velocities can be tuned by the particle aspect ratio. We hypothesize that the band gap originates from the primary eigenmode peak, the m-splitted (s,1,2) mode, of the particle resonating with the effective medium. These results reveal the potential for powerful control of the hypersonic phononic band diagram by combining anisotropic particles and self-assembly.

Dielectric spectroscopy of concentrated colloidal suspensions.

A comparison between experimental measurements and theoretical calculations of the permittivity and conductivity of concentrated colloidal suspensions is presented. Dielectric spectroscopy measurements for 100nm and 200nm diameter polystyrene spheres at volume fractions between ϕ=0.01-0.18 and electrolyte concentrations 0.01-1mM KCl (P.J. Beltramo, E.M. Furst, Langmuir 28 (2012) 10703-10712) are compared to cell-model calculations that account for the hydrodynamic and electrokinetic interactions between particles (F. Carrique, F.J. Arroyo, M.L. Jimenez, A.V. Delgado, J. Chem. Phys. 118 (2003) 1945-1956). Under most conditions, there is good agreement between experiment and theory. At low ionic strengths, the dielectric increment exhibits a low-frequency plateau in the experimental spectroscopy and cell model calculations. However, at the highest ionic strengths, the cell model predicts a low frequency plateau that is not observed experimentally. The conductivity increments qualitatively agree over all volume fractions, ionic strengths and frequencies.

Predicting the disorder-order transition of dielectrophoretic colloidal assembly with dielectric spectroscopy.

The dielectrophoretic assembly of colloidal suspensions into crystalline arrays is described by a master scaling that collapses the disorder-order transition as a function of field strength, frequency, and particle size. This master scaling has been verified for particle diameters ranging from 2a = 200 nm to 3 µm by light scattering (Lumsdon et al., Langmuir 2004, 20, 2108-2116; McMullan and Wagner, Langmuir 2012, 28, 4123-4130), optical laser tweezer measurements (Mittal et al., J. Chem. Phys. 2008, 129, 064513), and small-angle neutron scattering (McMullan and Wagner, Soft Matter 2010, 6, 5443-5450). In this work, we reconcile the empirical phase diagram with direct measurements of the colloid polarizability using dielectric spectroscopy. Dielectric spectroscopy confirms the origin of the order-disorder transition frequency dependence, including its quadratic scaling with particle radius, a(2), and provides an alternative method to search for optimal self-assembly conditions.

Transition from dilute to concentrated electrokinetic behavior in the dielectric spectra of a colloidal suspension.

Dielectric spectroscopy is used to measure the complex permittivity of 200 and 100 nm diameter polystyrene latex suspended in potassium chloride (KCl) solutions over the frequency range 10(4)-10(7) Hz as a function of particle volume fraction (ϕ) and ionic strength. Dilute suspension dielectric spectra are in excellent agreement with electrokinetic theory. A volume fraction dependence of the dielectric increment is observed for low electrolyte concentrations (0.01, 0.05, and 0.1 mM) above ϕ ≈ 0.02. This deviation from the dilute theory occurs at a critical frequency ω* that is a function of volume fraction, particle size, and ionic strength. The dielectric increment of suspensions at the highest salt concentration (1 mM) shows no volume fraction dependence up to ϕ = 0.09. Values of ω* are collapsed onto a master curve that accounts for the length and time scales of ion migration between neighboring particles. The measured conductivity increment is independent of volume fraction and agrees with theory after accounting for added counterions and nonspecific adsorption.

Dielectric spectroscopy of bidisperse colloidal suspensions.

Dielectric spectroscopy is used to measure the complex permittivity of bidisperse colloidal suspensions over the frequency range 2.5 kHz ≤ ω/2π ≤ 10 MHz using the spectrometer design of Hollingsworth and Saville (A.D. Hollingsworth, D.A. Saville, J. Colloid Interface Sci., 2003). Dielectric spectra of monodisperse polystyrene spheres of two diameters (530 nm and 1 µm) are fit to electrokinetic theory using the surface charge density as an adjustable parameter. Quantitative agreement is found in the dielectric increment and also for the conductivity increment, after considering the effect of added counterions and nonspecific adsorption. Bidisperse suspension spectra are a linear superposition of each particle's dielectric response. The results provide a simple method to extend standard electrokinetic theory based on a single particle size to dilute suspensions with many particle sizes and verify the sensitivity of the spectrometer.