We present a micropipet-assisted writing technique for formation of two-dimensional networks of phospholipid vesicles and nanotubes on functionalized and patterned substrates. The substrates are patterned with vesicle-adhesive circular spots (5−7.5 μm in diameter) consisting of a basal layer of biotin on gold and an apical coating of NeutrAvidin in a sandwich manner. The area surrounding the adhesive spots is coated with a phosphatidylcholine bilayer membrane, preventing protein and liposome adhesion. Networks were formed by aspirating a biotin-functionalized giant unilamellar or multilamellar liposome (5−50 μm in diameter) into a ∼3 μm inner diameter borosilicate glass micropipet. By using a pressurized-air microejection system, a portion of the liposome is then ejected back into the solution while forming a first vesicle ∼3 μm in diameter. This vesicle is placed on an adhesive spot. When the micropipet is moved, a nanotube connection is formed from the first vesicle and is pulled to the next adhesive spot where a second vesicle is ejected. This procedure can then be repeated until the lipid material is consumed in the pipet. The method allows for formation of networks with a large number of nodes and vertexes with well-defined geometry and surface adhesion, and represents a first step toward very large scale integration of nanotube−vesicle networks in, for example, nanofluidic applications.
Cell-cell communication is critical to the development, maintenance, and function of multicellular organisms. Classical mechanisms for intercellular communication include secretion of molecules into the extracellular space and transport of small molecules through gap junctions. Recent reports suggest that cells also can communicate over long distances via a network of transient intercellular nanotubes. Such nanotubes have been shown to mediate intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, intercellular nanotubes have been observed in vivo and have been shown to enhance the transmission of pathogens such as human immunodeficiency virus (HIV)-1 and prions in vitro. These studies indicate that intercellular nanotubes may play a role both in normal physiology and in disease.
Networks of nanotubes and vesicles offer a platform for construction of nanofluidic devices
operating on single molecule and particle level. Here one has the opportunity to study chemistry
in confined biomimetic compartments in an environment as close to nature as possible without
going in-vivo.
The development of lipid vesicle networks and the techniques involved has been an ongoing
process for over 10 years. Over the years weve expanded our abilities to observe, handle, and
predict nanotube vesicle networks. Understanding of the physical properties of these systems is
imperative to the explanation of the observed behavior in the conducted experiments. But were
quickly approaching the limit where we also require knowledge of how these systems act and
react to their environment. If we want to use these systems for anything more than just as cool
toy we need to get a grip on the hard physical aspects of how we choose to interact with these
systems.
The research in highly organized lipid vesicle networks is going into a regime where control of
surface properties becomes a fundamental interest. Without proper attention to the substrate we
will never achieve our scientific goals, the use of these systems in a user friendly, research or
medical industry environment, where they can be used in the research of biological membrane
behavior and cellular processes for the development of medical drugs.
The scientific field of lipid nanotube vesicle networks has taken several major steps toward a
technique that is of use for the general scientific community. Among these are the cell patterning
covered in paper I, concerning electroporation of cells, the pipette writing principle described in
paper II, and in paper III, the expansion of vesicle networks to the third dimension.
Nanofluidic devices are rapidly emerging as tools uniquely suited to transport and interrogate single molecules. We present a simple method to rapidly obtain compact surfactant nanotube networks of controlled geometry and length. The nanotubes, 100−300 nm in diameter, are pulled from lipid vesicles using a micropipet technique, with multilamellar vesicles serving as reservoirs of surfactant material. In a second step, the nanotubes are wired around microfabricated SU-8 pillars. In contrast to unrestrained surfactant networks that minimize their surface free energy by minimizing nanotube path length, the technique presented here can produce nanotube networks of arbitrary geometries. For example, nanotubes can be mounted directly on support pillars, and long stretches of nanotubes can be arranged in zigzag patterns with turn angles of 180°. The system is demonstrated to support electrophoretic transport of colloidal particles contained in the nanotubes down to the limit of single particles. We show that electrophoretic migration velocity is linearly dependent on the applied field strength and that a local narrowing of the nanotube diameter results from adhesion and bending around SU-8 pillars. The method presented here can aid in the fabrication of fully integrated and multiplexed nanofluidic devices that can operate with single molecules.
New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.
Surfactant lipids are an essential element of living cells. They are the basis for the biomembranes that envelope and divide cells into compartments. In addition to this static function, lipid membranes also play a role in dynamic processes such as transport and signaling.
The development of biomimetic lipid nanotube vesicle networks and the techniques involved has been an ongoing process for over 10 years. The techniques have expanded and our abilities to observe, handle, and predict nanotube vesicle network processes have increased. The applications of these systems range from the basic research of biological membrane behavior and cellular processes to the development of pharmaceutical drugs in a user friendly medical industry environment.
This thesis explores and expands techniques and applications of lipid nanotube vesicle mainly with a focus on immobilization and transport. Networks of nanotubes and vesicles offer a platform for construction of biomimetic nanofluidic devices operating down to single molecule and particle level. Highly organized and well defined lipid vesicle networks can be constructed with control over connectivity, container size, content, tube lengths and angle between nanotubes. Transport of fluid and particles confined in the network nodes can be controlled with several methods as well as modifications of content by controlled injections or chemical reaction dynamics.
Among these are the pipette writing principle described in paper I, allowing fast and efficient formation and immobilization of well defined networks with regard to size, geometry and connectivity. The method developed in paper II aid in the fabrication of fully integrated and multiplexed nanofluidic devices and expands the vesicle network connectivity to the third dimension. In paper III the use of electrophoretic transport show linear velocities of transported latex beads. Moreover it is proven that nanotubes adhered to a specific epoxy surface does not collapse and can sustain transport. Nanotubes wired to microfabricated substrates are shown to introduce new functionalities to vesicle networks. Based on the experimental observations and theoretical modeling in paper IV, we conclude that Y junctions observed in nanotube-vesicle networks forms by a zipper-like mechanism. Surfactants from two branches flow through the junction and form the extension of the third nanotube branch. The incorporation of an entirely biological component into the nanotube vesicle network in paper V not only shows proof of concept but also introduces new functionality to the system. The motile bacteria E. coli can be electroinjected into unilamellar lipid vesicles retaining both viability and motility. It is also suggested that they can be utilized to alter the chemical environment.
Methods based on self-assembly, self-organization, and forced shape transformations to form synthetic or semisynthetic enclosed lipid bilayer structures with several properties similar to biological nanocompartments are reviewed. The procedures offer unconventional micro- and nanofabrication routes to yield complex soft-matter devices for a variety of applications for example, in physical chemistry and nanotechnology. In particular, we describe novel micromanipulation methods for producing fluid-state lipid bilayer networks of nanotubes and surface-immobilized vesicles with controlled geometry, topology, membrane composition, and interior contents. Mass transport in nanotubes and materials exchange, for example, between conjugated containers, can be controlled by creating a surface tension gradient that gives rise to a moving boundary or by induced shape transformations. The network devices can operate with extremely small volume elements and low mass, to the limit of single molecules and particles at a length scale where a continuum mechanics approximation may break down. Thus, we also describe some concepts of anomalous fluctuation-dominated kinetics and anomalous diffusive behaviours, including hindered transport, as they might become important in studying chemistry and transport phenomena in these confined systems. The networks are suitable for initiating and controlling chemical reactions in confined biomimetic compartments for rationalizing, for example, enzyme behaviors, as well as for applications in nanofluidics, bioanalytical devices, and to construct computational and complex sensor systems with operations building on chemical kinetics, coupled reactions and controlled mass transport.
We investigate the formation of $\mathsf{Y}$ junctions in surfactant nanotubes connecting vesicles. Based on experimental observations of the surfactant flow on the nanotubes, we conclude that a $\mathsf{Y}$ junction propagates with a zipperlike mechanism. The surfactants from two nanotube branches undergo $1\ensuremath{\mathbin:}1$ mixing at the junction, and spontaneously form the extension of the third nanotube branch. Taking into account the tension driven surfactant flow, we develop a model for the $\mathsf{Y}$ junction dynamics that is in quantitative agreement with the experimental data.
