Generation of interconnected vesicles in a liposomal cell model

We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca2+ solution from the micropipette tip. IVs are rapidly and sequentially generated and inserted into the GUV interior and encapsulate portions of the micropipette fluid content. The IVs remain connected to the GUV membrane and are interlinked by short lipid nanotubes and resemble beads on a string. The vesicle chain-growth from the GUV membrane is maintained for as long as there is the supply of membrane material and Ca2+ solution, and the size of the individual IVs is controlled by the diameter of the micropipette tip. We also demonstrate that the IVs can be co-loaded with high concentrations of neurotransmitter and protein molecules and displaying a steep calcium ion concentration gradient across the membrane. These characteristics are analogous to native secretory vesicles and could, therefore, serve as a model system for studying secretory mechanisms in biological systems

A high-performance lab-on-a-chip liquid sensor employing surface acoustic wave resonance: part II

We recently introduced an in-liquid sensing concept based on surface acoustic resonance (SAR) in a lab-on-a-chip resonant device with one electrical port. The 185 MHz one-port SAR sensor has a sensitivity comparable to other surface acoustic wave (SAW) in-liquid sensors, while offering a high quality factor (Q) in water, low impedance, and fairly low susceptibility to viscous damping. In this work, we present significant design and performance enhancements of the original sensor presented in part I. A novel \u27lateral energy confinement\u27 (LEC) design is introduced, where the spatially varying reflectivity of the SAW reflectors enables strong SAW localization inside the sensing domain at resonance. An improvement in mass-sensitivity greater than 100% at resonance is achieved, while the measurement noise stays below 0.5 ppm. Sensing performance was evaluated through real-time measurements of the binding of 40 nm neutravidin-coated SiO2nanoparticles to a biotin-labeled lipid bilayer. Two complementary sensing parameters are studied, the shift of resonance frequency and the shift of conductance magnitude at resonance

Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%

Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment

A single nanoparticle platform has been developed through the modular and controlled layer-by-layer process to codeliver siRNA that knocks down a drug-resistance pathway in tumor cells and a chemotherapy drug to challenge a highly aggressive form of triple-negative breast cancer. Layer-by-layer films were formed on nanoparticles by alternately depositing siRNA and poly-l-arginine; a single bilayer on the nanoparticle surface could effectively load up to 3500 siRNA molecules, and the resulting LbL nanoparticles exhibit an extended serum half-life of 28 h. In animal models, one dose via intravenous administration significantly reduced the target gene expression in the tumors by almost 80%. By generating the siRNA-loaded film atop a doxorubicin-loaded liposome, we identified an effective combination therapy with siRNA targeting multidrug resistance protein 1, which significantly enhanced doxorubicin efficacy by 4 fold in vitro and led to up to an 8-fold decrease in tumor volume compared to the control treatments with no observed toxicity. The results indicate that the use of layer-by-layer films to modify a simple liposomal doxorubicin delivery construct with a synergistic siRNA can lead to significant tumor reduction in the cancers that are otherwise nonresponsive to treatment with Doxil or other common chemotherapy drugs. This approach provides a potential strategy to treat aggressive and resistant cancers, and a modular platform for a broad range of controlled multidrug therapies customizable to the cancer type in a singular nanoparticle delivery system.Janssen Pharmaceutical Ltd. (TRANSCEND Grant)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Health and Medical Research Council (Australia) (CJ Martin Fellowship)National Science Foundation (U.S.). Graduate Research FellowshipNatural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship

Lipid Nanotube Networks: Shape Transitions and Insights into the Dynamics of Self-Organization

Nanotube-vesicle networks (NVNs) are simplified models of cell membrane tubular systems which are dynamic transportation routs for molecular cargoes in biological cells. The presented work describes dynamic properties of NVNs such as self-organization, shape and topology transformations; moreover, specific geometric properties of the networks are used for controlling enzymatic reactions.A nanotube-vesicle network is a network of surface-adhered lipid vesicles (5-25 m in radius) connected by suspended lipid nanotubes (100-200 nm in radius). Vesicle size, nanotube length, and connectivity of a network can be controlled with high precision. Initially, the network is trapped in a high free energy state. By proper means, it is possible to trigger network self-organization towards a lower free energy state.Network evolution begins with merging of two adjacent nanotubes, and formation of a single nanotube three-way junction. Based on experimental observations of fluorescently labeled nanotubes and a theoretical model, the nanotube three-way junction is shown to propagate with a zipper-like mechanism, described in Paper I of this thesis. Lipids from two merging branches flow through the junction and form an extension on the third nanotube branch. Depending on the starting arrangement of the nanotubes, a NVN can evolve towards entangled and knotted geometries; or it can form a system of branching nanotubes. Paper II describes the formation of knotted nanotubes. The estimated size of the knot is comparable with the radius of a lipid nanotube. It is also demonstrated that such a knot can be used as a mechanical tweezer to capture and transport submicrometer-sized objects. In the experiments described in Paper III, NVNs are shown to form tree-like structures. The nanotubes arrange into symmetric three-way junctions with angles of 120o between the nanotubes. Moreover, the process of self-organization in the networks reveals a strong similarity with some optimization problems, such as the Euclidian Steiner Tree Problem. Paper IV suggests a method to form circular lipid nanotubes. The presented method gives new opportunities for preparing, manipulating and studying shape transitions of vesicles with non-spherical topology.Finally in Paper V, the geometry of a NVN is used to control the dynamics of an enzymatic reaction. Here, the vesicles are used as containers for reacting molecules, and the nanotubes serve as transportation routes. The narrow nanotube entrances act as transport barriers for the enzyme molecules. In such a reaction-diffusion system, the reaction occurs as a cascade through the containers and displays wave-like behavior

Ca2+ Gradient Induces Membrane Bending and Formation of Nanotubes in Giant Lipid Vesicles

Reshaping and bending of the cell membrane is imperative in many processes such as cell division, filopodia formation, and endocytosis. Understanding these shape transitions, will help to elucidate the underlying mechanisms of these essential cellular processes. In our work, we investigate an interplay between cell membrane morphology and chemical stimulation by constructing a biomimetic model system. More specifically, giant lipid vesicles were exposed to a chemical gradient of Ca2+, which was established over the membrane surface