Unnatural Peptide Assemblies Rapidly Deplete Cholesterol and Potently Inhibit Cancer Cells.

Cholesterol-rich membranes play a pivotal role in cancer initiation and progression, necessitating innovative approaches to target these membranes for cancer inhibition. Here we report the first case of unnatural peptide (1) assemblies capable of depleting cholesterol and inhibiting cancer cells. Peptide 1 self-assembles into micelles and is rapidly taken up by cancer cells, especially when combined with an acute cholesterol-depleting agent (MßCD). Click chemistry has confirmed that 1 depletes cell membrane cholesterol. It localizes in membrane-rich organelles, including the endoplasmic reticulum, Golgi apparatus, and lysosomes. Furthermore, 1 potently inhibits malignant cancer cells, working synergistically with cholesterol-lowering agents. Control experiments have confirmed that C-terminal capping and unnatural amino acid residues (i.e., BiP) are essential for both cholesterol depletion and potent cancer cell inhibition. This work highlights unnatural peptide assemblies as a promising platform for targeting the cell membrane in controlling cell fates.

Active Pulsatile Gels: From a Chemical Microreactor to a Polymeric Actuator.

We report on a synthesis protocol, experimental characterization, and theoretical modeling of active pulsatile Belousov-Zhabotinsky (BZ) hydrogels. Our two-step synthesis technique allows independent optimization of the geometry, the chemical, and the mechanical properties of BZ gels. We identify the role of the surrounding medium chemistry and gel radius for the occurrence of BZ gel oscillations, quantified by the Damköhler number, which is the ratio of chemical reaction to diffusion rates. Tuning the BZ gel size to maximize its chemomechanical oscillation amplitude, we find that its oscillatory strain amplitude is limited by the time scale of gel swelling relative to the chemical oscillation period. Our experimental findings are in good agreement with a Vanag-Epstein model of BZ chemistry and a Tanaka Fillmore theory of gel swelling dynamics.

Collective chemomechanical oscillations in active hydrogels.

We report on the collective response of an assembly of chemomechanical Belousov-Zhabotinsky (BZ) hydrogel beads. We first demonstrate that a single isolated spherical BZ hydrogel bead with a radius below a critical value does not oscillate, whereas an assembly of the same BZ hydrogel beads presents chemical oscillation. A BZ chemical model with an additional flux of chemicals out of the BZ hydrogel captures the experimentally observed transition from oxidized nonoscillating to oscillating BZ hydrogels and shows this transition is due to a flux of inhibitors out of the BZ hydrogel. The model also captures the role of neighboring BZ hydrogel beads in decreasing the critical size for an assembly of BZ hydrogel beads to oscillate. We finally leverage the quorum sensing behavior of the collective to trigger their chemomechanical oscillation and discuss how this collective effect can be used to enhance the oscillatory strain of these active BZ hydrogels. These findings could help guide the eventual fabrication of a swarm of autonomous, communicating, and motile hydrogels.

A machine learning approach to robustly determine director fields and analyze defects in active nematics.

Active nematics are dense systems of rodlike particles that consume energy to drive motion at the level of the individual particles. They exist in natural systems like biological tissues and artificial materials such as suspensions of self-propelled colloidal particles or synthetic microswimmers. Active nematics have attracted significant attention in recent years due to their spectacular nonequilibrium collective spatiotemporal dynamics, which may enable applications in fields such as robotics, drug delivery, and materials science. The director field, which measures the direction and degree of alignment of the local nematic orientation, is a crucial characteristic of active nematics and is essential for studying topological defects. However, determining the director field is a significant challenge in many experimental systems. Although director fields can be derived from images of active nematics using traditional imaging processing methods, the accuracy of such methods is highly sensitive to the settings of the algorithms. These settings must be tuned from image to image due to experimental noise, intrinsic noise of the imaging technology, and perturbations caused by changes in experimental conditions. This sensitivity currently limits automatic analysis of active nematics. To address this, we developed a machine learning model for extracting reliable director fields from raw experimental images, which enables accurate analysis of topological defects. Application of the algorithm to experimental data demonstrates that the approach is robust and highly generalizable to experimental settings that are different from those in the training data. It could be a promising tool for investigating active nematics and may be generalized to other active matter systems.

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids.

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T = 3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose three-dimensional geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures.

Light-activated microtubule-based two-dimensional active nematic.

We assess the ability of two light responsive kinesin motor clusters to drive dynamics of microtubule-based active nematics: opto-K401, a processive motor, and opto-K365, a non-processive motor. Measurements reveal an order of magnitude improvement in the contrast of nematic flow speeds between maximally- and minimally-illuminated states for opto-K365 motors when compared to opto-K401 construct. For opto-K365 nematics, we characterize both the steady-state flow and defect density as a function of applied light. We also examine the transient behavior as the system switches between steady-states upon changes in light intensities. Although nematic flows reach a steady state within tens of seconds, the defect density exhibits transient behavior for up to 10 minutes, showing a separation between small-scale active flows and system-scale structural states. Our work establishes an experimental platform that can exploit spatiotemporally-heterogeneous patterns of activity to generate targeted dynamical states.

From disks to channels: dynamics of active nematics confined to an annulus.

Confinement can be used to systematically tame turbulent dynamics occurring in active fluids. Although periodic channels are the simplest geometries to study confinement numerically, the corresponding experimental realizations require closed racetracks. Here, we computationally study 2D active nematics confined to such a geometry-an annulus. By systematically varying the annulus inner radius and channel width, we bridge the behaviors observed in the previously studied asymptotic limits of the annulus geometry: a disk and an infinite channel. We identify new steady-state behaviors, which reveal the influence of boundary curvature and its interplay with confinement. We also show that, below a threshold inner radius, the dynamics are insensitive to the presence of the inner hole. We explain this insensitivity through a simple scaling analysis. Our work sheds further light on design principles for using confinement to control the dynamics of active nematics.

Geometrically programmed self-limited assembly of tubules using DNA origami colloids.

Self-assembly is one of the most promising strategies for making functional materials at the nanoscale, yet new design principles for making self-limiting architectures, rather than spatially unlimited periodic lattice structures, are needed. To address this challenge, we explore the tradeoffs between addressable assembly and self-closing assembly of a specific class of self-limiting structures: cylindrical tubules. We make triangular subunits using DNA origami that have specific, valence-limited interactions and designed binding angles, and we study their assembly into tubules that have a self-limited width that is much larger than the size of an individual subunit. In the simplest case, the tubules are assembled from a single component by geometrically programming the dihedral angles between neighboring subunits. We show that the tubules can reach many micrometers in length and that their average width can be prescribed through the dihedral angles. We find that there is a distribution in the width and the chirality of the tubules, which we rationalize by developing a model that considers the finite bending rigidity of the assembled structure as well as the mechanism of self-closure. Finally, we demonstrate that the distributions of tubules can be further sculpted by increasing the number of subunit species, thereby increasing the assembly complexity, and demonstrate that using two subunit species successfully reduces the number of available end states by half. These results help to shed light on the roles of assembly complexity and geometry in self-limited assembly and could be extended to other self-limiting architectures, such as shells, toroids, or triply periodic frameworks.

Pattern formation in a four-ring reaction-diffusion network with heterogeneity.

In networks of nonlinear oscillators, symmetries place hard constraints on the system that can be exploited to predict universal dynamical features and steady states, providing a rare generic organizing principle for far-from-equilibrium systems. However, the robustness of this class of theories to symmetry-disrupting imperfections is untested in free-running (i.e., non-computer-controlled) systems. Here, we develop a model experimental reaction-diffusion network of chemical oscillators to test applications of the theory of dynamical systems with symmeries in the context of self-organizing systems relevant to biology and soft robotics. The network is a ring of four microreactors containing the oscillatory Belousov-Zhabotinsky reaction coupled to nearest neighbors via diffusion. Assuming homogeneity across the oscillators, theory predicts four categories of stable spatiotemporal phase-locked periodic states and four categories of invariant manifolds that guide and structure transitions between phase-locked states. In our experiments, we observed that three of the four phase-locked states were displaced from their idealized positions and, in the ensemble of measurements, appeared as clusters of different shapes and sizes, and that one of the predicted states was absent. We also observed the predicted symmetry-derived synchronous clustered transients that occur when the dynamical trajectories coincide with invariant manifolds. Quantitative agreement between experiment and numerical simulations is found by accounting for the small amount of experimentally determined heterogeneity in intrinsic frequency. We further elucidate how different patterns of heterogeneity impact each attractor differently through a bifurcation analysis. We show that examining bifurcations along invariant manifolds provides a general framework for developing intuition about how chemical-specific dynamics interact with topology in the presence of heterogeneity that can be applied to other oscillators in other topologies.

Extensile to contractile transition in active microtubule-actin composites generates layered asters with programmable lifetimes.

We study a reconstituted composite system consisting of an active microtubule network interdigitated with a passive network of entangled F-actin filaments. Increasing the concentration of filamentous actin controls the emergent dynamics, inducing a transition from turbulent-like flows to bulk contractions. At intermediate concentrations, where the active stresses change their symmetry from anisotropic extensile to isotropic contracting, the composite separates into layered asters that coexist with the background turbulent fluid. Contracted onion-like asters have a radially extending microtubule-rich cortex that envelops alternating layers of microtubules and F-actin. These self-regulating structures undergo internal reorganization, which appears to minimize the surface area and maintain the ordered layering, even when undergoing aster merging events. Finally, the layered asters are metastable structures. Their lifetime, which ranges from minutes to hours, is encoded in the material properties of the composite. These results challenge the current models of active matter. They demonstrate self-organized dynamical states and patterns evocative of those observed in the cytoskeleton do not require precise biochemical regulation, but can arise from purely mechanical interactions of actively driven filamentous materials.

Multiscale Microtubule Dynamics in Active Nematics.

In microtubule-based active nematics, motor-driven extensile motion of microtubule bundles powers chaotic large-scale dynamics. We quantify the interfilament sliding motion both in isolated bundles and in a dense active nematic. The extension speed of an isolated microtubule pair is comparable to the molecular motor stepping speed. In contrast, the net extension in dense 2D active nematics is significantly slower; the interfilament sliding speeds are widely distributed about the average and the filaments exhibit both contractile and extensile relative motion. These measurements highlight the challenge of connecting the extension rate of isolated bundles to the multimotor and multifilament interactions present in a dense 2D active nematic. They also provide quantitative data that is essential for building multiscale models.

Effects of confinement on the dynamics and correlation scales in kinesin-microtubule active fluids.

We study the influence of solid boundaries on dynamics and structure of kinesin-driven microtubule active fluids as the height of the container, H, increases from hundreds of micrometers to several millimeters. By three-dimensional tracking of passive tracers dispersed in the active fluid, we observe that the activity level, characterized by velocity fluctuations, increases as system size increases and retains a small-scale isotropy. Concomitantly, as the confinement level decreases, the velocity-velocity temporal correlation develops a strong positive correlation at longer times, suggesting the establishment of a "memory". We estimate the characteristic size of the flow structures from the spatial correlation function and find that, as the confinement becomes weaker, the correlation length, l_{c}, saturates at approximately 400 microns. This saturation suggests an intrinsic length scale which, along with the small-scale isotropy, demonstrates the multiscale nature of this kinesin-driven bundled microtubule active system.

Submersed micropatterned structures control active nematic flow, topology, and concentration.

Coupling between flows and material properties imbues rheological matter with its wide-ranging applicability, hence the excitement for harnessing the rheology of active fluids for which internal structure and continuous energy injection lead to spontaneous flows and complex, out-of-equilibrium dynamics. We propose and demonstrate a convenient, highly tunable method for controlling flow, topology, and composition within active films. Our approach establishes rheological coupling via the indirect presence of fully submersed micropatterned structures within a thin, underlying oil layer. Simulations reveal that micropatterned structures produce effective virtual boundaries within the superjacent active nematic film due to differences in viscous dissipation as a function of depth. This accessible method of applying position-dependent, effective dissipation to the active films presents a nonintrusive pathway for engineering active microfluidic systems.

Programmable icosahedral shell system for virus trapping.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.

Partition, Reaction, and Diffusion Coefficients of Bromine in Elastomeric Polydimethylsiloxane.

Experiments and models were used to determine the extent to which aqueous bromine permeated into, and reacted with, the elastomer polydimethylsiloxane (PDMS). Thin films of PDMS were immersed in bromine water, and the absorbance of bromine in the aqueous phase was measured as a function of time. Kinetics were studied as a function of mass and thickness of the immersed PDMS films. We attribute the decrease of bromine in solution to permeation into PDMS, followed by a combination of diffusion, reversible binding, and an irreversible reaction with PDMS. In order to decouple the irreversible reaction from the reversible processes, kinetics were also studied for bromine-passivated PDMS films. Fits of the models to a variety of experiments yielded the partition coefficient of bromine between the water and PDMS phases, the diffusion constant of bromine in PDMS, the irreversible reaction constant between bromine and PDMS, the molar concentration of the reactive sites within PDMS, and the on and off rates of reversible binding of bromine to PDMS. Developing a quantitative reaction-diffusion model accounting for the transport of bromine through PDMS is necessary for the design of microfluidic devices fabricated using PDMS, which are used in experimental studies of the nonlinear dynamics of reaction-diffusion networks containing Belousov-Zhabotinsky chemical oscillators.

Membrane Remodeling by DNA Origami Nanorods: Experiments Exploring the Parameter Space for Vesicle Remodeling.

Inspired by the ability of cell membranes to alter their shape in response to bound particles, we report an experimental study of long, slender nanorods binding to lipid bilayer vesicles and altering the membrane shape. Our work illuminates the role of particle concentration, adhesion strength, and membrane tension in determining the membrane morphology. We combined giant unilamellar vesicles with oppositely charged nanorods, carefully tuning the adhesion strength, membrane tension, and particle concentration. With increasing adhesion strength, the primary behaviors observed were membrane deformation, vesicle-vesicle adhesion, and vesicle rupture. These behaviors were observed in well-defined regions in the parameter space with sharp transitions between them. We observed the deformation of the membrane resulting in tubulation, textured surfaces, and small and large lipid-particle aggregates. These responses are robust and repeatable and provide a new physical understanding of the dependence on the shape, binding affinity, and particle concentration in membrane remodeling. The design principles derived from these experiments may lead to new bioinspired membrane-based materials.

An Arduino-based constant pressure fluid pump.

Constant pressure pumps are an invaluable yet underutilized resource for microfluidic flow systems. In particular, constant pressure pumps are able to stabilize the fluid pressure in systems where the viscosity may change due to chemical reactions or the flow rate may vary due to deformations of the channels. The constant pressure pump presented here is designed on the premise of creating and maintaining a pressure differential between the laboratory and a pressure reservoir. This pressure reservoir is then used to drive the input fluid at the specified gauge pressure. The pump design presented here is perfect for primarily undergraduate institutions and other laboratories with modest research budgets as it can be built for under US$100 and construction is within the scope of an advanced undergraduate. The pump consists of an Arduino-compatible microcontroller, Adafruit electronic components, low-voltage air pump, Nalgene water bottle, and various fluid components. A complete parts list is included in the appendix. Comparable commercial pumps have a retail price in excess of US$5000. Multiple pump designs were constructed and tested with the ability to hold a constant pressure of up to 14 psig (97 kPa-gauge) with a maximum flow rate of 65 [Formula: see text]L/s for water.

Machine learning forecasting of active nematics.

Active nematics are a class of far-from-equilibrium materials characterized by local orientational order of force-generating, anisotropic constitutes. Traditional methods for predicting the dynamics of active nematics rely on hydrodynamic models, which accurately describe idealized flows and many of the steady-state properties, but do not capture certain detailed dynamics of experimental active nematics. We have developed a deep learning approach that uses a Convolutional Long-Short-Term-Memory (ConvLSTM) algorithm to automatically learn and forecast the dynamics of active nematics. We demonstrate our purely data-driven approach on experiments of 2D unconfined active nematics of extensile microtubule bundles, as well as on data from numerical simulations of active nematics.

Impact of PDMS-Based Microfluidics on Belousov-Zhabotinsky Chemical Oscillators.

Sub-nanoliter volumes of the Belousov-Zhabotinsky (BZ) reaction are sealed in microfluidic devices made from polydimethylsiloxane (PDMS). Bromine, which is a BZ reaction intermediate that participates in the inhibitory pathway of the reaction, is known to permeate into PDMS, and it has been suggested that PDMS and bromine can react ( J. Phys. Chem. A. 108, 2004, 1325-1332). We characterize the extent to which PDMS affects BZ oscillations by varying the volume of the PDMS surrounding the BZ reactors. We measure how the oscillation period varies with PDMS volume and compare with a theoretical reaction-diffusion model, concluding that bromine reacts with PDMS. We demonstrate that minimizing the amount of PDMS by making the samples as thin as possible maximizes the number of oscillations before the BZ reaction reaches equilibrium and ceases to oscillate. We also demonstrate that the deleterious effects of the PDMS-BZ interactions are somewhat mitigated by imposing constant chemical boundary conditions through using a light-sensitive catalyst, ruthenium, in combination with patterned illumination. Furthermore, we show that light can modulate the frequency and phase of the BZ oscillators contained in a PDMS matrix by 20-30%.

Optimal Control of Active Nematics.

In this work we present the first systematic framework to sculpt active nematic systems, using optimal control theory and a hydrodynamic model of active nematics. We demonstrate the use of two different control fields, (i) applied vorticity and (ii) activity strength, to shape the dynamics of an extensile active nematic that is confined to a disk. In the absence of control inputs, the system exhibits two attractors, clockwise and counterclockwise circulating states characterized by two co-rotating topological +1/2 defects. We specifically seek spatiotemporal inputs that switch the system from one attractor to the other; we also examine phase-shifting perturbations. We identify control inputs by optimizing a penalty functional with three contributions: total control effort, spatial gradients in the control, and deviations from the desired trajectory. This work demonstrates that optimal control theory can be used to calculate nontrivial inputs capable of restructuring active nematics in a manner that is economical, smooth, and rapid, and therefore will serve as a guide to experimental efforts to control active matter.