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.

Membrane Tension–Mediated Growth of Liposomes

“…We produced GUVs (≈10 µm in diameter) using OLA . For observing a clean batch of GUVs, we modified the previously reported method to separate the liposomes from the waste product (1‐octanol droplets), while still using density differences as the basis of separation: First, we punched a large collection well (4 mm in diameter) at the end of the production channel in order to collect GUVs at its bottom and let the waste product (1‐octanol droplets) float to the top of the fluid‐filled well. Next, we encapsulated dextran (molecular weight ( M W ) = 6000) inside the GUVs that served a dual purpose: i) It induced sedimentation of the GUVs at the bottom of the well by making them denser than the environment (Figure b); ii) It acted as an osmolyte to induce the necessary membrane tension.…”

“…Next, we encapsulated dextran (molecular weight ( M W ) = 6000) inside the GUVs that served a dual purpose: i) It induced sedimentation of the GUVs at the bottom of the well by making them denser than the environment (Figure b); ii) It acted as an osmolyte to induce the necessary membrane tension. Along with a population‐level analysis which allowed us to obtain data from large population sizes ( n > 1000), we also tracked individual GUVs using a previously published separation device, with an additional array of physical traps after the separation module, in order to immobilize purified GUVs.…”

“…In order to capture the growth process at a single‐liposome level, we utilized a more sophisticated setup involving microfluidic traps. These physical traps were integrated into a design used for on‐chip density‐based separation of liposomes from 1‐octanol droplets, right after the separation hole. A feeding channel was used to subsequently flow the SUV‐containing solution along the traps.…”

Mechanical Division of Cell-Sized Liposomes

“…So far, it was not possible to significantly modulate the flow velocity, since the splitter is directly connected to the production junction. Using a recently developed density-based liposome separation technique which decouples the production and further downstream experimentation, 33 it will be possible to vary the collision velocity of the liposomes, and investigate its effect on the splitting efficiency. Finally, combining the physical division with a growth module to achieve a growth-division cycle will be a significant step in the context of synthetic biology.…”

Biophysical characterization reveals the similarities of liposomes produced using microfluidics and electroformation

“…In the field of synthetic biology, mechanical division (20) and membrane tension mediated growth (21) of OLA vesicles, as well as coacervate formation within liposomes have been shown (22). Furthermore, protocols for the purification of OLA liposomes have been presented (23).…”