The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.
Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (>10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 µL), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.
All cells have a plasma membrane as their physical boundary, and this membrane is essentially a scaffold in the form of a lipid bilayer formed by the self-assembly of amphiphilic lipid molecules. Liposomes are the minimal synthetic counterparts of biological cells; they have an aqueous lumen surrounded by phospholipids, which form a lipid bilayer with the hydrophilic head groups facing the aqueous phase and the hydrophobic tails buried inward. The stability of liposomes is governed by the hydrophobic effect, as well as the hydrophilicity between the polar groups, van der Waals forces between the hydrophobic carbon tails, and the hydrogen bonding between water molecules and the hydrophilic heads1,2. Depending on the number of lipid bilayers, liposomes can be classified into two main categories, namely, unilamellar vesicles comprising a single bilayer and multilamellar vesicles constructed from multiple bilayers. Unilamellar vesicles are further classified based on their sizes. Typically spherical in shape, they can be produced in a variety of sizes, including small unilamellar vesicles (SUV, 30-100 nm diameter), large unilamellar vesicles (LUV, 100-1,000 nm diameter), and finally, giant unilamellar vesicles (GUV, >1,000 nm diameter)3,4. Various techniques have been developed to produce liposomes, and these can be categorized broadly into bulk techniques5 and microfluidic techniques6. Commonly practiced bulk techniques include lipid film rehydration, electroformation, inverted emulsion transfer, and extrusion7,8,9,10. These techniques are relatively simple and effective, and these are the prime reasons for their widespread usage in the synthetic biology community. However, at the same time, they suffer from major drawbacks with regard to the polydispersity in size, the lack of control over the lamellarity, and low encapsulation efficiency7,11. Techniques like continuous droplet interface crossing encapsulation (cDICE)12 and droplet shooting and size filtration (DSSF)13 overcome these limitations to some extent.
Microfluidic approaches have been rising to prominence over the last decade. Microfluidic technology provides a controllable environment for manipulating fluid flows within user-defined microchannels owing to the characteristic laminar flow and diffusion-dominated mass transfer. The resulting lab-on-a-chip devices offer unique possibilities for the spatiotemporal control of molecules, with significantly reduced sample volumes and multiplexing capabilities14. Numerous microfluidic methods to make liposomes have been developed, including pulsed jetting15, double emulsion templating16, transient membrane ejection17, droplet emulsion transfer18, and hydrodynamic focusing19. These techniques produce monodispersed, unilamellar, cell-sized liposomes with high encapsulation efficiency and high throughput.
This article details the procedure for octanol-assisted liposome assembly (OLA), an on-chip microfluidic method based on the hydrodynamic pinch-off and subsequent solvent dewetting mechanism20 (Figure 1). One can relate the working of OLA to a bubble-blowing process. A six-way junction focuses the inner aqueous (IA) phase, two lipid-carrying organic (LO) streams, and two surfactant-containing outer aqueous (OA) streams at a single spot. This results in water-in-(lipids + octanol)-in-water double emulsion droplets. As these droplets flow downstream, interfacial energy minimization, external shear flow, and interaction with the channel walls lead to the formation of a lipid bilayer at the interface as the solvent pocket becomes detached, thus forming unilamellar liposomes. Depending on the size of the octanol pocket, the dewetting process can take tens of seconds to a couple of minutes. At the end of the exit channel, the less dense octanol droplets float to the surface, whereas the heavier liposomes (due to a denser encapsulated solution) sink to the bottom of the visualization chamber ready for experimentation. As a representative experiment, the process of liquid-liquid phase separation (LLPS) inside liposomes is demonstrated. For that, the required components are encapsulated inside liposomes at an acidic pH that prevents LLPS. By externally triggering a pH change and, thus, a transmembrane proton flux, phase-separated condensate droplets are formed inside the liposomes. This highlights the effective encapsulation and manipulation capabilities of the OLA system.
1. Fabricating the master wafer
2. Preparing the microfluidic device
3. Making the PDMS-coated glass slide
4. Bonding of the microfluidic device
5. Surface functionalization of the microfluidic device
NOTE: Prior to surface functionalization, it is important to calibrate the pressure pump as per the manufacturer's protocol (see Table of Materials) and assemble the tubing to connect it to the microfluidic device.
6. Octanol-assisted liposome assembly (OLA)
This study demonstrates the formation of membraneless condensates via the process of liquid-liquid phase separation (LLPS) inside liposomes as a representative experiment.
Sample preparation
The IA, OA, ES, and feed solution (FS) are prepared as follows:
IA: 12% glycerol, 5 mM dextran, 150 mM KCl, 5 mg/mL poly-L-lysine (PLL), 0.05 mg/mL poly-L-lysine-FITC labeled (PLL-FITC), 8 mM adenosine triphosphate (ATP), 15 mM citrate-HCl (pH 4)
OA: 12% v/v glycerol, 5% w/v F68, 150 mM KCl, 15 mM citrate-HCl (pH 4)
ES: 12% glycerol, 150 mM KCl, 15 mM citrate-HCl (pH 4)
FS: 12% glycerol, 150 mM KCl, 75 mM Tris-HCl (pH 9)
Condensate formation inside liposomes
A simple assay of pH-sensitive complex coacervation of positively charged poly-L-lysine (PLL) and negatively charged multivalent adenosine triphosphate (ATP) was selected to demonstrate the phenomena of LLPS in liposomes. To prevent phase separation of polylysine and ATP during encapsulation, the pH of the solution was maintained at 4, at which ATP is neutral. Increasing the pH of the ES by adding FS (a buffer of pH 9) eventually increased the pH inside the liposomes due to transmembrane proton flux, making ATP negatively charged and triggering its phase separation with positively charged PLL21 (Figure 4A). After about 2 h of liposome generation, the IA and LO pressures were switched off. The OA channel pressure was kept at 100 mbar to flow the remaining liposomes into the observation chamber slowly. Once all the liposomes in the channel were recovered in the EW, the pressures were switched off to stop the flow and prevent the liposomes from moving. The liposomes generated at a lower pH showed a homogeneous fluorescence (from the encapsulated fluorescent PLL-FITC) in their lumen (Figure 4B,D). Octanol droplets floating at the surface were removed by carefully pipetting out 5 µL of solution from the top to prevent them from affecting further pipetting steps. Subsequently, 10 µL of FS buffer was added to the EW, which induced phase separation of the encapsulated PLL and ATP. The homogeneous FITC fluorescence from each liposome gradually transformed into distinct fluorescent condensate droplets. Eventually, the individual droplets merged into one bigger condensate droplet that freely diffused within the liposomes (Figure 4C,E–G).
Figure 1: Schematic showing the assembly and working of OLA. The pressure controller is connected to the reservoirs containing outer aqueous, lipid-in-octanol, and inner aqueous solutions. The tubes inserted into the reservoirs are connected to the respective inlets of the OLA device. Appropriate flows in the three channels lead to the formation of water-in-(lipid-in-octanol)-in-water double emulsions. The formed double emulsions migrate to the exit well, during which the octanol pockets detach to form liposomes. The formed liposomes are collected at the bottom of the well for visualization and further experimentation. Please click here to view a larger version of this figure.
Figure 2: Preparation of the OLA chip (photolithography, microfabrication, surface treatment). (A) Digital design showing the key features of the OLA design, including three inlets, an outlet, and a six-way production junction. (B) A schematic of the master wafer showing multiple OLA designs produced using UV lithography. (C) A PDMS elastomer cast on the master wafer, placed in a well created out of aluminum foil, and cured by baking at 70 °C for 2 h. (D) A microfluidic device bonded using oxygen plasma treatment, where the PDMS block containing the OLA design is attached to a PDMS-coated glass slide. (E) Surface functionalization of the fabricated chip to make the device partially hydrophilic. This is done by flowing 5% w/v PVA for 5 min from the outer aqueous channel toward the exit channel. Positive air pressure in the other channels prevents the PVA solution from entering these channels. (F) PVA is removed by applying a vacuum in the exit channel. The device is baked at 120 °C for 15 min and is then ready to use. Please click here to view a larger version of this figure.
Figure 3: Demonstration of OLA efficiently producing monodispersed double emulsions and eventually liposomes with excellent encapsulation. (A) A bright-field image showing rapid generation of double-emulsion droplets. (B) The fluorescent lipid channel shows the formation of an octanol pocket due to partial dewetting. The lipid-in-octanol phase contained a mixture of DOPC (5 mg/mL) and Lis Rhod PE at a 1,000:1 ratio. (C) The inner aqueous channel showing encapsulation of yellow fluorescent protein (YFP). The insets in (A–C) show representative zoomed-in views of the respective panels (scale bars = 10 µm). (D–F) Dewetting of the octanol pocket in the exit channel, which forms a liposome. Please click here to view a larger version of this figure.
Figure 4: pH-triggered liquid-liquid phase separation of pLL/ATP within liposomes. (A) Schematic of the pH-dependent transitioning of the homogenous solution of pLL and ATP encapsulated within the liposome (left) to phase-separated pLL/ATP coacervates (right). The initial acidic environment in the liposome renders the molecular charge of ATP to be neutral, inhibiting coacervation. When the pH inside the liposomes equilibrates with the externally applied pH increase, ATP gains a negative charge, triggering coacervation. (B–C) Line graphs (corresponding to the dotted lines in panels [D] and [G], respectively) showing the spatial distribution of pLL (green channel) and the membrane (red channel). (D–G) Time-lapse images showing the formation of pLL/ATP coacervates within the liposomes. The external addition of a basic buffer raises the pH level inside the liposomes over the course of minutes and initiates coacervation. t = 0 min refers to the time just before the occurrence of the first coacervation event. Please click here to view a larger version of this figure.
Supplementary Coding File 1: CAD file of the OLA design. Please click here to download this File.
Cellular complexity makes it extremely difficult to understand living cells when studied as a whole. Reducing the redundancy and interconnectivity of cells by reconstituting the key components in vitro is necessary to further our understanding of biological systems and create artificial cellular mimics for biotechnological applications22,23,24. Liposomes serve as an excellent minimal system to understand cellular phenomena. A non-exhaustive list of these phenomena includes cytoskeleton dynamics and resulting membrane deformations25,26, spatiotemporal regulation of biomolecular condensates27,28 and their interaction with the membrane29, encapsulation of a wide variety of biomolecules, including cell-free transcription-translation systems30, cell-free lipid synthesis31, and evolution of proteins32,33,34. Liposomes have also been widely used as carriers for drug delivery and have been approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for clinical usage11. Liposomes and lipid-based nanoparticles are used as carriers for drug delivery, including mRNA vaccines like the recent COVID-19 vaccine35.
This study describes a detailed protocol on photolithography, microfluidic assembly, and surface functionalization to perform the OLA technique in order to generate on-chip liposomes (Figure 1 and Figure 3). The liposomes produced using OLA are monodispersed (coefficient of variation: 4%-11% of the mean) and in the biological cell-sized range (typically between 5-50 µm in diameter), and they can be tuned using appropriate flow velocities of the inner and outer aqueous channels36. For example, the liposome population seen in Figure 1 has a size distribution of 23.3 µm ± 1.8 µm (n = 50). With typical production rates of 10-30 Hz, the formed liposomes are unilamellar, as was previously confirmed by inserting a single bilayer-specific transmembrane protein, alpha-hemolysin36, into the membrane, as well as by using the dithionate-bleaching assay37. OLA is also compatible with a wide variety of lipid compositions, offering the user flexibility in the choice of lipids. Importantly, the initially used lipid composition is reflected in the final liposome composition38.
Being a relatively new technology, there is further scope for improving OLA. For instance, surface functionalization using PVA is crucial but tedious to perform. An easier and simpler way to surface-functionalize the device would significantly reduce the chip fabrication time as well as chip-to-chip variability. Pluronic F68 surfactant is an important component in the outer aqueous phase to initially stabilize the double emulsions; nonetheless, its usage can be restrictive in certain cases. Replacing Pluronic F68 with a more biocompatible surfactant or complete surfactant removal may improve the versatility of the system. During the migration of generated double emulsions in the exit channel, they can burst due to shear. Upgrading the OLA design to improve the double emulsion stability and octanol pocket separation could, thus, increase the throughput. Nonetheless, OLA has several advantages, which mainly include the efficient encapsulation, monodispersed and unilamellar liposomes, and controlled on-chip experimentation.
OLA has been employed and adapted in a diverse range of studies, including growth and division of liposomes39, studying the process of liquid-liquid phase separation27 and its interaction with the membrane21, and understanding bacterial growth bioproduct formation in liposomes40. OLA-based high-throughput assays are also being used to understand ion transport across the membrane41 and drug permeability across the lipid bilayer37, as a drug delivery system for therapeutic purposes42, to study the effect of antimicrobials on membrane43, and as a tool to encapsulate liquid crystals44. In addition to the wide range of applications of OLA technology, modified versions of OLA suited for particular purposes are being developed45,46,47,48,49,50. Overall, considering the pros and cons, we strongly believe that OLA is a versatile platform for synthetic biology.
The authors have nothing to disclose.
We would like to acknowledge Dolf Weijers, Vera Gorelova, and Mark Roosjen for kindly providing us with YFP. S.D. acknowledges financial support from the Dutch Research Council (grant number: OCENW.KLEIN.465).
1-Octanol | Sigma-Aldrich | No. 297887 | |
1.5 mL tubes | Fisher scientific | 10451043 | Eppendorf 3810X Polypropylene microcentrifuge tubes |
ATP | Sigma-Aldrich | No. A2383 | |
Biopsy punch | Darwin microfluidics | PT-T983-05 | 0.5 mm and 3 mm diameter |
Citrate-base | Sigma-Aldrich | No. 71405 | |
Dextran | Sigma-Aldrich | No. 31388 | Mr~6,000 |
Direct-write optical lithography machine | Durham Magneto Optics Ltd | MicroWriter ML3 Baby | setup and software |
DOPC lipid | Avanti | SKU:850375C | |
F68 | Sigma-Aldrich | No. 24040032 | |
Glass cover slip | Corning | #1, 24 x 40 mm | |
Glycerol | Sigma-Aldrich | No. G2025 | |
Hydrochloric acid | Thermo Scientific Acros | No. 124630010 | |
Liss Rhod PE lipid | Avanti | SKU:810150C | |
Parafilm | Sigma-Aldrich | No. P7793 | |
Photoresist | Micro resist technology GmbH | EpoCore 10 | |
Photoresist developer | micro resist technology GmbH | mr-Dev 600 | |
Plasma cleaner | Harrick plasma | PDC-32G | |
Polydimethylsiloxane | Dow | Sylgard 184 | PDMS and curing agent |
Poly-L-lysine | Sigma-Aldrich | No. P7890 | |
Poly-L-lysine–FITC Labeled | Sigma-Aldrich | No. P3543 | |
Polyvinyl alcohol | Sigma-Aldrich | no. P8136 | molecular weight 30,000–70,000, 87%–90% hydrolyzed |
Pressure controller | Elveflow | OBK1 Mk3+ | Flow controller |
Scotch tape | Magic Tape Invisible Matt Tape | ||
Silicon wafer | Silicon Materials | 0620R16002 | |
Spin coater | Laurell Technologies Corporation | Model WS-650MZ-23NPP | |
Stainless Steel 90° Bent PDMS Couplers | Darwin microfluidics | PN-BEN-23G | |
Tris-base | Sigma-Aldrich | No. 252859 | |
Tygon tubing | Darwin microfluidics | 1/16" OD x 0.02" ID | |
UV laser | 365 nm wavelength |