Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation

Abstract

Encapsulation of macromolecules within lipid vesicles has the potential to drive biological discovery and enable development of novel, cell-like therapeutics and sensors. However, rapid and reliable production of large numbers of unilamellar vesicles loaded with unrestricted and precisely-controlled contents requires new technologies that overcome size, uniformity, and throughput limitations of existing approaches. Here we present a high-throughput microfluidic method for vesicle formation and encapsulation using an inkjet printer at rates up to 200 Hz. We show how multiple high-frequency pulses of the inkjet's piezoelectric actuator create a microfluidic jet that deforms a bilayer lipid membrane, controlling formation of individual vesicles. Variations in pulse number, pulse voltage, and solution viscosity are used to control the vesicle size. As a first step toward cell-like reconstitution using this method, we encapsulate the cytoskeletal protein actin and use co-encapsulated microspheres to track its polymerization into a densely entangled cytoskeletal network upon vesicle formation.

Forming giant vesicles with controlled membrane composition, asymmetry, and contents

“…Planar lipid bilayers can be created at the interface between two aqueous droplets that are initially surrounded by oil (containing dissolved lipids) and then brought into contact, an approach pioneered by Bayley et al to study membrane pores (29). We have previously demonstrated that these planar bilayers can be deformed by microfluidic jetting with a piezoelectric inkjet nozzle to form giant unilamellar vesicles (26). However, our previous use of lipids dissolved in oil prevented the inclusion of physiologically important signaling lipids.…”

“…A first step toward engineering systems that recapitulate the physical boundary conditions of cells is encapsulation of components in giant vesicles of controlled lipid chemistry. Several recent microfluidic techniques (22,(24)(25)(26)(27) have achieved formation of giant vesicles with controlled contents using oil-water interfaces to define lipid membranes. However, these techniques form the membranes from lipids dissolved in oil and are incompatible with many biologically important lipids that display poor solubility in oil due to their net charge or saturated fatty acid tails.…”

“…We begin by demonstrating incorporation of physiologically relevant signaling lipids into giant unilamellar vesicles (GUVs) made by microfluidic jetting (26,27), which enables simultaneous control of internal contents. We extend this approach to form GUVs with asymmetric lipid bilayers and then demonstrate incorporation and orientation of transmembrane proteins in the GUVs.…”

Easy-to-Assemble and Adjustable Coaxial Flow-Focusing Microfluidic Device for Generating Controllable Water/Oil/Water Double Emulsions: Toward Templating Giant Liposomes with Different Properties

“…However, the as formed vesicles often lack monodispersity and/or the product yield is relative low. More recently, new techniques such as pulsed jet flow [17,18] and, with the development of soft lithographic methods [19], microfluidic devices have been applied in the formation of lipid vesicles. In particular, microfluidics, i.e., the manipulation of fluids on the micrometric scale, has proven an excellent technique for generating double emulsion for GUV encapsulation.…”

Artificial membranes for membrane protein purification, functionality and structure studies

“…The size of liposomes can also be tuned by subsequent procedures such as swelling [17], extrusion [18], emulsion, sonication [19], electroformation [20], inkjet formation [21], microfluidic jet [22,23] freeze thaw cycles [24]. Sonication and extrusion are the two most popular techniques.…”