Unilamellar vesicle formation and encapsulation by microfluidic jetting

Abstract

Compartmentalization of biomolecules within lipid membranes is a fundamental requirement of living systems and an essential feature of many pharmaceutical therapies. However, applications of membrane-enclosed solutions of proteins, DNA, and other biologically active compounds have been limited by the difficulty of forming unilamellar vesicles with controlled contents in a repeatable manner. Here, we demonstrate a method for simultaneously creating and loading giant unilamellar vesicles (GUVs) using a pulsed microfluidic jet. Akin to blowing a bubble, the microfluidic jet deforms a planar lipid bilayer into a vesicle that is filled with solution from the jet and separates from the planar bilayer. In contrast with existing techniques, our method rapidly generates multiple monodisperse, unilamellar vesicles containing solutions of unrestricted composition and molecular weight. Using the microfluidic jetting technique, we demonstrate repeatable encapsulation of 500-nm particles into GUVs and show that functional pore proteins can be incorporated into the vesicle membrane to mediate transport. The ability of microfluidic jetting to controllably encapsulate solutions inside of GUVs creates new opportunities for the study and use of compartmentalized biomolecular systems in science, industry, and medicine.

Fig. 1.

Formation of GUVs by microfluidic jetting. (a) Schematic of the piezoelectric-driven microfluidic jetting device assembled with the planar lipid bilayer chamber. (b) Close-up schematic of the vesicle-formation process, highlighting the interaction of a vortex ring structure with the planar lipid bilayer. (c) Independent, monodisperse, GUVs resulting from pulsed microfluidic jetting. (Scale bar: 100 μm.) (d) Consistency of vesicle diameter over seven separate trials including a total of 46 GUVs. Error bars represent the first standard deviation.

Fig. 2.

Vesicle-formation process. Individual frames taken from a high-speed movie (7,500 fps) of vesicle formation (t1–t8 correspond to 667 μs, 800 μs, 1,067 μs, 1,467 μs, 2,000 μs, 2,267 μs, 3,200 μs, 4,667 μs after the start of actuator expansion). Bright-field contrast is created by jetting a 200 mM sucrose solution into a surrounding solution of 200 mM glucose. (Scale bar: 100 μm.)

Fig. 3.

Kinematics of vortex ring–membrane collision. Decay of vortex ring velocity and membrane protrusion velocity in time. Power law curve fits were applied beginning at the time of maximum velocity. For the vortex (red circles), V = 0.59t^−1.26 (R² = 0.98). For the protrusion (blue squares), V = 0.26t^−2.00 (R² = 0.93). (Inset) Bright-field image of a vortex ring created in the absence of the planar bilayer using an actuator expansion rate equivalent to that used to form GUVs. (Scale bar: 50 μm.)

Fig. 4.

Characterization of GUVs formed by microfluidic jetting. (a) Membrane labeling and volume exclusion for the same GUV. (Scale bars: 50 μm.) (a Left) Phase contrast image of the GUV. (a Center) Labeling of the GUV membrane using BODIPY dye. (a Right) Wide-field fluorescence image documenting exclusion of sulforhodamine B dye by the GUV. (b) Encapsulation of polystyrene beads into GUVs with the microfluidic jet. (b Left) Bright-field image of four GUVs created from a single planar bilayer. (b Right) Encapsulation of fluorescent beads (500 nm diameter, FITC) in GUVs.

Fig. 5.

Protein pore-mediated transport of solutes across vesicle boundaries. (a) Schematic diagram. (b) Experimental results showing that a GUV initially excluding FITC dye increases in fluorescence relative to the fluorescence of the external solution after addition of α-hemolysin. α-Hemolysin is added to 2.5 μg/ml at time 0, and the vesicle is tracked for 104 min, at which point relative fluorescence has reached 76%.