Name: on-chip octanol-assisted liposome assembly for bioengineering
Uploaded: 2023-03-17T13:55:00
Description: Watch this Scientific Journal Video about On-Chip Octanol-Assisted Liposome Assembly for Bioengineering at JoVE.com

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. Fabricating the master wafer
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>To obtain a 10 &#181;m thic...

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...

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, &#62;1,000 nm diameter)3,4. Various techniques have been developed to produce liposomes, and these can be categorized broadly into bulk techniques5&#160;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&#160;and droplet shooting and size filtration (DSSF)13&#160;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&#160;(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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Octanol</td>
			<td>Sigma-Aldrich</td>
			<td>No. 297887</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Fisher scientific</td>
			<td>10451043</td>
			<td>Eppendorf 3810X Polypropylene microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>No. A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Darwin microfluidics</td>
			<td>PT-T983-05</td>
			<td>0.5 mm and 3 mm diameter</td>
		</tr>
		<tr>
			<td>Citrate-base</td>
			<td>Sigma-Aldrich</td>
			<td>No. 71405</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>No. 31388</td>
			<td>Mr~6,000</td>
		</tr>
		<tr>
			<td>Direct-write optical lithography machine</td>
			<td>Durham Magneto Optics Ltd</td>
			<td>MicroWriter ML3 Baby</td>
			<td>setup and software</td>
		</tr>
		<tr>
			<td>DOPC lipid</td>
			<td>Avanti</td>
			<td>SKU:850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>F68</td>
			<td>Sigma-Aldrich</td>
			<td>No. 24040032</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slip</td>
			<td>Corning</td>
			<td>#1, 24 x 40 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>No. G2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Thermo Scientific Acros</td>
			<td>No. 124630010</td>
			<td></td>
		</tr>
		<tr>
			<td>Liss Rhod PE lipid</td>
			<td>Avanti</td>
			<td>SKU:810150C</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>No. P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>Micro resist technology GmbH</td>
			<td>EpoCore 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist developer</td>
			<td>micro resist technology GmbH</td>
			<td>mr-Dev 600</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane</td>
			<td>Dow</td>
			<td>Sylgard 184</td>
			<td>PDMS and curing agent</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>No. P7890</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine&#8211;FITC Labeled</td>
			<td>Sigma-Aldrich</td>
			<td>No. P3543</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>no. P8136</td>
			<td>molecular weight 30,000&#8211;70,000, 87%&#8211;90% hydrolyzed</td>
		</tr>
		<tr>
			<td>Pressure controller</td>
			<td>Elveflow&#160;</td>
			<td>OBK1 Mk3+</td>
			<td>Flow controller</td>
		</tr>
		<tr>
			<td>Scotch tape</td>
			<td>Magic Tape Invisible Matt Tape</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Silicon Materials</td>
			<td>0620R16002</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater&#160;</td>
			<td>Laurell Technologies Corporation</td>
			<td>Model WS-650MZ-23NPP</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel 90&#176; Bent PDMS Couplers</td>
			<td>Darwin microfluidics</td>
			<td>PN-BEN-23G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma-Aldrich</td>
			<td>No. 252859</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Darwin microfluidics</td>
			<td>1/16&#34; OD x 0.02&#34; ID</td>
			<td></td>
		</tr>
		<tr>
			<td>UV laser&#160;</td>
			<td></td>
			<td></td>
			<td>365 nm wavelength</td>
		</tr>
	</tbody>
</table>

on-chip octanol-assisted liposome assembly for bioengineering

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 (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), 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.

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)<...

Watch this Scientific Journal Video about On-Chip Octanol-Assisted Liposome Assembly for Bioengineering at JoVE.com

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering (Video) | JoVE

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.

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are ...

Research

JoVE Journal

Bioengineering

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