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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Fabricating the master wafer

  1. Take a 4 in (10 cm) diameter clean silicon wafer (see Table of Materials). Clean it further using pressurized air to remove any dust particles.
  2. Mount the wafer on a spin coater, and gently dispense ~5 mL of a negative photoresist (see Table of Materials) in the center of the wafer. Try to avoid air bubbles, as they might interfere with the downstream printing process of the wafer.
  3. To obtain a 10 µm thick photoresist layer, spin-coat the wafer at 500 rpm for 30 s with an acceleration of 100 rpm/s for initial spreading, followed by a 60 s spin at 3,000 rpm with an acceleration of 500 rpm/s. In case a different thickness is desired, change the spinning parameters according to the manufacturer's instructions.
  4. Bake the wafer on a heating plate for 2 min at 65 °C and then for 5 min at 95 °C.
  5. Once the wafer cools down, mount the wafer in the printing chamber of the direct-write optical lithography machine (see Table of Materials), and feed the OLA design (Figure 2A, Supplementary Coding File 1) into the software.
    NOTE: The OLA design essentially consists of two OA channels, two LO channels, and one IA channel, which merge to form a six-way junction that continues as a post-production channel and ends up in the experimental well (EW).
  6. Print the OLA design(s) on the coated wafer using a UV laser (see Table of Materials) with a dose of 300 mJ/cm2.
  7. Once the design is printed, bake the wafer at 65 °C for 1 min, followed by 95 °C for 3 min.
  8. At this point, ensure that the outline of the printed device appears on the wafer and is visible to the naked eye. To wash off the uncured photoresist, dip the wafer in a glass beaker containing the developer solution (see Table of Materials) until the uncured photoresist is fully removed.
    NOTE: Excess developer treatment can affect the design resolution.
  9. Wash the wafer with acetone, isopropanol, and deionized (DI) water in sequence, and finally, with pressurized air/N2 using a blow gun.
  10. Hard-bake the wafer at 150 °C for 30 min to ensure that the printed design is firmly attached to the wafer surface and does not come off in the downstream fabrication process. The master wafer is then ready for further use (Figure 2B).

2. Preparing the microfluidic device

  1. Place the master wafer on a square piece of aluminum, and wrap the aluminum foil around the wafer, forming a well-like structure (Figure 2C).
  2. Measure 40 g of polydimethylsiloxane (PDMS) and 4 g of curing agent (10:1, weight ratio, see Table of Materials) in a 50 mL centrifuge tube, and mix vigorously using a spatula or a pipette tip for 2-3 min.
  3. The vigorous mixing of the PDMS and curing agent traps air bubbles inside the mixture. Spin the tube at 100 x g for 2-3 min to remove majority of large air bubbles.
  4. Pour approximately 15-20 g of the mixture prepared in step 2.3 over the master wafer, and de-gas using a desiccator. Save the excess mixture to make PDMS-coated glass slides (step 3).
  5. Incubate the assembly in an oven at 70 °C for at least 2 h.
  6. Take the master wafer out of the oven and let it cool down. To remove the solidified PDMS block, remove the aluminum foil, and carefully peel off the PDMS block from the edge of the wafer.
    NOTE: The wafer is fragile and might break, so it is important to do this process carefully.
  7. Once the PDMS block is removed, keep the patterned structure facing upward, and using a sharp blade or a scalpel, cut individual PDMS blocks, each containing a single microfluidic device.
  8. Place the PDMS block on a dark surface, and adjust the light direction (a table lamp comes in handy for this) so that the engraved channels are shiny and, as a result, visible. Make holes in the inlets and the exit channel using biopsy punches of 0.5 mm and 3 mm diameter (see Table of Materials), respectively.
    NOTE: Use a sharp biopsy punch to avoid cracks in the PDMS, which can cause leakage later. Gently push the biopsy punch through the PDMS block, and ensure it passes completely through it. To remove the biopsy punch, gently retract it while rotating it in alternate directions. The PDMS block with the engraved OLA design is now ready to bind to a PDMS-coated glass slide.

3. Making the PDMS-coated glass slide

  1. Take a transparent glass coverslip, pour approximately 0.5 mL of PDMS (prepared in excess in step 2.4) onto the center of the glass slide, and spread it across the coverslip by gently tilting the glass slide, ensuring total coverage of the glass slide with PDMS.
  2. Mount the glass slide on the spin coater, ensure its centrally placed (so that the middle of the slide overlaps with the center of the pressure shaft), and spin the glass slide at 500 rpm for 15 s (at an increment of 100 rpm/s) and then at 1,000 rpm for 30 s (at an increment of 500 rpm/s).
  3. Place the PDMS-coated glass slide (coated side facing upward) on a raised platform like a block of PDMS (1 cm x 1 cm) in a covered Petri dish, and bake it at 70 °C for 2 h.

4. Bonding of the microfluidic device

  1. Clean the PDMS block (prepared in step 2) by sticking and removing commercially available scotch tape (see Table of Materials) twice. Ensure to keep the cleaned side facing up until use.
  2. Clean the PDMS-coated glass slide with pressurized air/N2 using a blow gun, and keep the clean side facing up.
  3. Place the PDMS block (engraved channels facing up) and the PDMS-coated glass slide (coated side facing up) in the vacuum chamber of the plasma cleaner (see Table of Materials).
  4. Switch on the vacuum, and expose the contents to air plasma at a radio frequency of 12 MHz (RF mode high) for 15 s to activate the surfaces. Oxygen plasma can be seen in the form of a pinkish hue.
  5. Immediately after the plasma treatment, place the PDMS-coated glass slide on a clean surface with the PDMS side facing upward. Gently place the PDMS block with the microfluidic pattern now facing toward the PDMS-coated glass slide, allowing them to bond (Figure 2D).
    NOTE: Removing the air trapped in the device is crucial to ensure thorough bonding.
  6. Bake the bonded devices at 70 °C for 2 h.

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.

  1. Connecting the microfluidic device to the pressure pumps
    1. Connect the pressure-driven flow controller to an external pressure source (up to 6 bars). At least four independent air pressure channels are required: three individual modules of 0-2 bar, and one module of −0.9-4 bar.
    2. Calibrate the pressure pump according to the manufacturer's protocol (see Table of Materials).
    3. Cut the tubing (1/16 in OD x 0.02 in ID) into four equal-sized pieces approximately 20 cm in length.
    4. Insert 23 G stainless steel 90° bent connectors (see Table of Materials) at one end of each piece. This end is later inserted into the three inlets (OA, LO, and IA) of the microfluidic device and the fourth one is used to remove excess solution (step 5.2.5).
    5. Insert the other end of the tubing into the microfluidic reservoir stand, and seal it using microfluidic fittings, ensuring the tubing is long enough to touch the bottom of the reservoir (1.5 mL tubes, see Table of Materials). This prevents unwanted air bubbles from entering the microfluidic device during the PVA treatment/liposome production.
  2. PVA treatment of the exit channel
    NOTE: Prior to liposome generation, it is crucial to render the device partially hydrophilic downstream of the production junction. This is done by treating these channels with 5% (w/v) polyvinyl alcohol (PVA) solution. The PVA solution is prepared by dissolving the PVA powder (see Table of Materials) in water at 80 °C for 3 h with constant stirring using a magnetic stirrer. Filter the solution prior to using it for surface treatment. Immediately after the bonding of the device, the microfluidic channels are hydrophilic due to the plasma-induced surface activation, and they will gradually become hydrophobic again. It is recommended to wait for 2 h after the baking (step 4.6) before starting the PVA treatment.
    1. Dispense 200 µL of 5% w/v PVA solution into a 1.5 mL tube, and connect it to the microfluidic reservoir stand. Insert the tubing (the one mentioned in step 5.1.3) such that one end is submerged in the PVA solution and the other end is connected to the inlet of the OA channel of the microfluidic device.
      NOTE: A lower PVA concentration can lead to sub-standard surface functionalization.
    2. Repeat the above step without PVA (empty 1.5 mL tube), and connect it to the LO and IA channels.
    3. Increase the pressure of the OA phase to 100 mbar to flow the PVA solution in the OA channels. Increase the pressures of the IA and LO phases to 120 mbar in order to prevent the backflow of PVA solution inside these channels (Figure 2E).
      NOTE: If needed, adjust the pressures of the individual channels in order to keep the meniscus steady at the production junction.
    4. Flow the PVA solution in this manner for approximately 5 min, ensuring complete functionalization of the exit channel.
    5. To remove the PVA solution, detach the tubing from the OA inlet, and immediately increase the pressure in the LO and IA channels to 2 bar. Simultaneously, use a tubing connected to a negative pressure channel (−900 mbar) remove excess PVA first from the exit channel and then from the OA inlet (Figure 2F).
    6. Bake the device at 120 °C for 15 min, and let it cool down before use. The device can be stored under ambient conditions for at least 1 month.
      NOTE: It is recommended to wait for 1 day (at room temperature) before proceeding with OLA to ensure the untreated PDMS regains its hydrophobicity after the plasma treatment.

6. Octanol-assisted liposome assembly (OLA)

  1. Preparing the lipid stock
    NOTE: Here, a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Liss Rhod PE) lipids (see Table of Materials) are used as an example; users should prepare the lipid composition they need in a similar manner.
    1. Dispense 76 µL of DOPC (25 mg/mL) and 16.6 µL of Liss Rhod PE (1 mg/mL) into a round-bottom flask using separate glass syringes.
    2. Keep the round-bottom flask upright, and use a compressed N2 blow gun to give a gentle stream of nitrogen to evaporate the chloroform and form a dried lipid film at the bottom of the flask.
    3. Place the flask in the desiccator for at least 2 h under a continuous vacuum to remove any remaining chloroform.
    4. Add 100 µL of ethanol into the round-bottom flask, followed by gentle pipetting or shaking to ensure the lipids are dissolved to form a 10% (w/v) lipid stock.
      NOTE: In case a particular lipid composition is not completely soluble in ethanol, use an ethanol/chloroform mixture, keeping the volume of chloroform as small as possible.
    5. Pipette the solution into a dark glass vial. Gently flush the vial with nitrogen using a blow gun to replace the air with an inert atmosphere. Seal the lid with paraffin film, and store at −20 °C.
      NOTE: The stock solution can be used for up to a few months. Each time the vial is opened, gently replace the air with nitrogen, and reseal with lid with paraffin film.
  2. Preparing the OLA solutions
    1. Make stock solutions of the indispensable components: 20 mM dextran (Mw 6,000); 60% (v/v) glycerol; a buffer of choice. In this case, 5x phosphate-buffered saline (0.68 M NaCl, 13.5 mM KCl, 50 mM Na2HPO4, 9 mM KH2PO4; pH 7.4) was prepared (see Table of Materials).
      NOTE: As pure glycerol is highly viscous, it is recommended to cut out a small piece at the end of the pipette tip in a slanted manner for effective pipetting.
    2. Prepare 100 µL of IA, OA, and exit solution (ES, the desired solution to fill the EW) samples. For ease of visualization, yellow fluorescent protein (YFP) was added to the IA solution in this particular case. Check the osmotic balance between the IA and OA by calculating the osmolarity of the encapsulated components and adding an appropriate amount of sugar or salt into the OA if needed. This is done in order to prevent the bursting or shrinking of the liposomes during production.
      NOTE: The final compositions of the three phases are as follows:
      IA: 15% glycerol, 5 mM dextran (Mw 6,000), 5.4 µM YFP, 1x PBS
      OA: 15% glycerol, 5% F68 surfactant, 1x PBS
      ​ES: 15% glycerol, 1x PBS
      A lower concentration of F68 surfactant negatively affects the generation of double emulsions.
    3. Prepare 80 µL of the LO phase by pipetting 4 µL of stock lipid into 76 µL of 1-octanol (see Table of Materials). The final concentration of DOPC is 5 mg/mL.
    4. Centrifuge the samples at 700 x g for 60 s to remove any large aggregates before proceeding with the microfluidic experiments. This prevents any obvious clogging factors from entering the microfluidic chip.
  3. Liposome production
    1. Dispense the solutions (IA, LO, OA) into three 1.5 mL tubes, assemble them (as mentioned in step 5.1), and connect them to the PVA-treated microfluidic chip (step 5.2.6).
    2. Apply a positive pressure on the three channels: ~100 mbar on the IA and LO channels and ~200 mbar on the OA channel.
      NOTE: Since the OA pressure is higher than the others, the OA solution will be the first to arrive at EW; this is highly recommended.
    3. Once the OA solution reaches EW, decrease the OA pressure to 100 mbar and increase the LO to 200 mbar.  The LO solution arriving at the production junction will likely result in air bubbles because of air being displaced from the LO channels. Once the air bubbles are dispensed into the EW, adjust the LO pressure to 100 mbar. Lastly, increase the IA pressure to 200 mbar, and wait until all the air in the IA channel is removed. The ideal sequence of arrival at the junction of the three phases is, thus, OA, LO, and IA.
    4. After removing the air from the three channels, adjust all the channel pressures to 50 mbar and pipette 10 µL of ES into the exit chamber. After pipetting the ES, put a cover slide on the exit hole to avoid evaporation. In case the LO is pushed back, gradually increase the LO pressure in 1 mbar increments until it starts to flow to the junction.
    5. Once all three phases start co-flowing at the junction, ensure double emulsion production begins, and adjust according to its quality (Figure 3A-C). Change the pressures gradually (steps of 0.1-1 mbar) rather than abruptly unless the pressure is being changed to eliminate an unwanted clog in the channel.
      NOTE: The channels to adjust depends on what is happening at the junction. For example, the IA needs to be decreased if the emulsions are too big; the OA needs to be increased if double emulsions form but burst instead of getting pinched off; and the LO needs to be decreased if the octanol pocket is too big.
    6. Check that the double-emulsion droplets flow downstream to EW. As they flow, octanol pockets become more and more prominent and finally get pinched off, forming liposomes (Figure 3D-F). Ensure that the post-production channel is long enough and that the migration of the double emulsions is slow enough for dewetting.
      NOTE: Within minutes after decent production, liposomes and octanol droplets will exit the post-production channel and go into the EW. As a result, being less dense than water, the octanol droplets float to the surface of the ES. Due to the addition of dextran in the IA, the liposomes are heavier than their surroundings and go to the bottom of the chamber ready for observation and further manipulation.

Results

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)<...

Discussion

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 no...

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

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).

Materials

NameCompanyCatalog NumberComments
1-OctanolSigma-AldrichNo. 297887
1.5 mL tubesFisher scientific10451043Eppendorf 3810X Polypropylene microcentrifuge tubes
ATPSigma-AldrichNo. A2383
Biopsy punchDarwin microfluidicsPT-T983-050.5 mm and 3 mm diameter
Citrate-baseSigma-AldrichNo. 71405
DextranSigma-AldrichNo. 31388Mr~6,000
Direct-write optical lithography machineDurham Magneto Optics LtdMicroWriter ML3 Babysetup and software
DOPC lipidAvantiSKU:850375C
F68Sigma-AldrichNo. 24040032
Glass cover slipCorning#1, 24 x 40 mm
GlycerolSigma-AldrichNo. G2025
Hydrochloric acidThermo Scientific AcrosNo. 124630010
Liss Rhod PE lipidAvantiSKU:810150C
ParafilmSigma-AldrichNo. P7793
PhotoresistMicro resist technology GmbHEpoCore 10
Photoresist developermicro resist technology GmbHmr-Dev 600
Plasma cleanerHarrick plasmaPDC-32G
PolydimethylsiloxaneDowSylgard 184PDMS and curing agent
Poly-L-lysineSigma-AldrichNo. P7890
Poly-L-lysine–FITC LabeledSigma-AldrichNo. P3543
Polyvinyl alcoholSigma-Aldrichno. P8136molecular weight 30,000–70,000, 87%–90% hydrolyzed
Pressure controllerElveflow OBK1 Mk3+Flow controller
Scotch tapeMagic Tape Invisible Matt Tape
Silicon waferSilicon Materials0620R16002
Spin coater Laurell Technologies CorporationModel WS-650MZ-23NPP
Stainless Steel 90° Bent PDMS CouplersDarwin microfluidicsPN-BEN-23G
Tris-baseSigma-AldrichNo. 252859
Tygon tubingDarwin microfluidics1/16" OD x 0.02" ID
UV laser 365 nm wavelength

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