On-chip density-based purification of liposomes

Abstract

Due to their cell membrane-mimicking properties, liposomes have served as a versatile research tool in science, from membrane biophysics and drug delivery systems to bottom-up synthetic cells. We recently reported a novel microfluidic method, Octanol-assisted Liposome Assembly (OLA), to form cell-sized, monodisperse, unilamellar liposomes with excellent encapsulation efficiency. Although OLA provides crucial advantages over alternative methods, it suffers from the presence of 1-octanol droplets, an inevitable by-product of the production process. These droplets can adversely affect the system regarding liposome stability, channel clogging, and imaging quality. In this paper, we report a density-based technique to separate the liposomes from droplets, integrated on the same chip. We show that this method can yield highly pure (>95%) liposome samples. We also present data showing that a variety of other separation techniques (based on size or relative permittivity) were unsuccessful. Our density-based separation approach favourably decouples the production and separation module, thus allowing freshly prepared liposomes to be used for downstream on-chip experimentation. This simple separation technique will make OLA a more versatile and widely applicable tool.

FIG. 1.

On-chip density-based separation of 1-octanol droplets from OLA-based liposomes. (a) Side-view schematic of the device, showing the technique leading to the separation of droplets from liposomes. The double-emulsion droplets produced at the junction give rise to liposomes and 1-octanol droplets, which coexist in the pre-hole channel. As they enter the separation hole, the droplets drift upwards, while the liposomes are slowly sucked into the post-hole channel. (b) Fluorescence image of the pre-hole channel showing individual liposomes and droplets, and double-emulsion droplets on the verge of budding-off. The droplet marked by a white arrow is an example of a larger 1-octanol droplet produced by an instability at the junction. (c) Fluorescence image of the post-hole channel predominantly showing liposomes and some very small droplets (indicated by white arrow).

FIG. 2.

Efficiency of the density-based separation technique. (a) Purity of liposomes obtained at different post-hole pressures. The pre-hole purity is also shown for comparison. The purity obtained is an average of at least three separate runs (number of particles analysed for each data set is >1500). Error bars indicate standard deviations. (b) An example showing the frequency histograms of the liposomes in pre-hole (magenta, n = 1028) and post-hole (cyan, n = 541) channels. (c) Example of the size distribution of droplets in the pre-hole channel (n = 2195). One can clearly see three discrete populations, belonging to—from left to right—smaller satellite droplets, droplets that are a result of the budding-off process from the liposomes, and bigger droplets that are formed as a result of instability at the production junction. The latter two populations (liposomes >3.9 μm) make up more than 95% of the population. (d) Example of the size distribution of the contaminating droplets in the post-hole channel (n = 540), after the separation takes place. More than 86% of the droplets are below 3.9 μm, the cut-off value used for the purity analysis (see main text).

FIG. 3.

DLD and PFF did not achieve efficient liposome-droplet separation. (a) 1-octanol droplets aggregated at the supporting pillars (striped circles) before the DLD array (top, fluorescence image) and even more in the DLD array, destroying its functionality (bottom, bright-field image). The arrows indicate prominent aggregates. Increasing the flow rates in the DLD array stalled the liposome production. (b) Very low number of liposomes (only those with the 1-octanol pocket still attached) could be separated from droplets using PFF (dashed line acts as a guideline and white arrow indicates the flow direction). Virtually, every liposome burst due to the high flow rates in the pinched section. Decreasing the flow rate in the pinched section, to prevent bursting, led to an effectively zero separation efficiency. (c) Top-view schematic showing the main problem of implementing DLD and PFF to separate droplets and liposomes, namely, the incompatible flow requirements. The liposome production using OLA needs low flow rates, but the DLD and PFF methods require higher flow rates and additional sheath flows. Due to the physical connection between the production module and the module with the separation method, it becomes impossible to satisfy both requirements, leading to the problems displayed in the other panels.

FIG. 4.

Diverting the path of water-in-octanol droplets using DEP. (a) The DEP junction consists of a low-resistance (top) and a high-resistance (bottom) channel. If the voltage on the electrodes (the black channels) is off, the droplets follow the low-resistance path. (b) Once the voltage is turned on (300 V, 5 kHz), the dielectrophoretic force diverts the droplets into the high-resistance channel at the bottom. Two droplets are encircled (in green and in red) at consecutive time-frames, as a guide to the eye. The arrows indicate the flow direction. The time difference between consecutive frames is 100 ms. Note that this technique worked well for water droplets in 1-octanol but was unsuccessful for 1-octanol droplets in water.