Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2024 Sep 26.
doi: 10.1039/d4lc00380b. Online ahead of print.

Microfluidic technologies for lipid vesicle generation

Yu Cheng  1   2 , Callum D Hay  1   2 , Suchaya M Mahuttanatan  1   2 , James W Hindley  1   2 , Oscar Ces  1   2 , Yuval Elani  1   3
Affiliations
Review

Microfluidic technologies for lipid vesicle generation

Yu Cheng et al. Lab Chip. .

Abstract

Encapsulating biological and non-biological materials in lipid vesicles presents significant potential in both industrial and academic settings. When smaller than 100 nm, lipid vesicles and lipid nanoparticles are ideal vehicles for drug delivery, facilitating the delivery of payloads, improving pharmacokinetics, and reducing the off-target effects of therapeutics. When larger than 1 μm, vesicles are useful as model membranes for biophysical studies, as synthetic cell chassis, as bio-inspired supramolecular devices, and as the basis of protocells to explore the origin of life. As applications of lipid vesicles gain prominence in the fields of nanomedicine, biotechnology, and synthetic biology, there is a demand for advanced technologies for their controlled construction, with microfluidic methods at the forefront of these developments. Compared to conventional bulk methods, emerging microfluidic methods offer advantages such as precise size control, increased production throughput, high encapsulation efficiency, user-defined membrane properties (i.e., lipid composition, vesicular architecture, compartmentalisation, membrane asymmetry, etc.), and potential integration with lab-on-chip manipulation and analysis modules. We provide a review of microfluidic lipid vesicle generation technologies, focusing on recent advances and state-of-the-art techniques. Principal technologies are described, and key research milestones are highlighted. The advantages and limitations of each approach are evaluated, and challenges and opportunities for microfluidic engineering of lipid vesicles to underpin a new generation of therapeutics, vaccines, sensors, and bio-inspired technologies are presented.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Lipids and vesicles. Top panel: Lipid self-assembly into vesicles is driven by the hydrophobic effect, minimising the interactions between hydrophobic tails and aqueous solution. Bottom panel: Typical lipid structure (POPC) is shown on the left. Common hydrophilic head groups of lipids and their charges at a physiological pH are listed. The hydrophobic tails of lipids can be saturated or unsaturated. For instance, the POPC lipid has one saturated 16 : 0 chain (16 carbons and 0 double bonds) and one unsaturated 18 : 1 chain (18 carbons and 1 double bond). 9 and 10 on the unsaturated tail of POPC are the carbons, between which the double bond locates.
Fig. 2
Fig. 2. Schematic representation of the dominant microfluidic platforms for preparing various lipid vesicles. Vesicles with diameters smaller than 100 nm are described as ‘small’ or ‘nano’, this includes small unilamellar vesicles (SUVs) and lipid nanoparticles (LNPs). Vesicles with diameters between 100 nm and 1 μm are described as ‘large’. Vesicles with diameters larger than 1 μm are described as ‘giant’, including giant unilamellar vesicles (GUVs), vesosomes (vesicle-in-vesicle), multilamellar vesicles (MLVs) and multicompartmental vesicles (MCVs). Microfluidic platforms represented by micromixers (reproduced from ref. with permission from the American Society of Gene & Cell Therapy, copyright [2012]) and MHF (reproduced from ref. with permission from Springer Nature, copyright [2016]) have demonstrated great potential in preparing lipid vesicles with nanoscale sizes for medical applications. Emulsion-based microfluidics focuses on preparing giant liposomal products as cell models or bioreactors from water-in-oil (W/O) emulsions. The pulsed jetting method (reproduced from ref. with permission from the American Chemical Society, copyright [2007]) can prepare vesicles of ‘small’, ‘large’, and ‘giant’ sizes.
Fig. 3
Fig. 3. Schematic representations of drug-loaded lipid vesicles (left) and nucleic acid-loaded lipid nanoparticles (LNPs)/lipoplexes (right). For drug-loaded vesicles, different types of drug molecules can be loaded through different mechanisms. For active targeting and controlled release, ligands can be attached. The dashed line indicates that vesicle surfaces can be modified to be neutral, negative, or positive; not a mix of charges on the same vesicle. For the delivery of nucleic acids, LNPs (left half) are inverted micelles whose inner cores are occupied by cationic or ionizable lipids, which are usually formed by passive loading, while lipoplexes (right half) retain the continuous bilayer structure of their precursor liposomes, which are usually formed by active loading.
Fig. 4
Fig. 4. Microfluidic hydrodynamic focusing. a| (i) Schematic of liposome formation through microfluidic hydrodynamic focusing. Two aqueous streams focus one lipid organic stream. Reproduced from ref. with permission from the American Chemical Society, copyright [2004]. Numerical simulations comparing ethanol concentration profiles within MHF (ii) and VFF (iii) systems. In the VFF system (not to scale), its microchannel aspect ratio is 1000 : 1, much larger than 0.5 : 1 in the conventional MHF system. Reproduced from ref. with permission from John Wiley and Sons, copyright [2015]. b| Schematic of capillary focusing liposome formation device (not to scale). A lipid alcohol solution is continuously injected into the intra-annular capillary tubing and hydrodynamically focused in three dimensions by an exterior sheath flow of aqueous buffer from a surrounding glass multi-capillary array. Reproduced from ref. with permission from the Royal Society of Chemistry, copyright [2014]. c| Microfluidic vortex focusing (MVF) device design and operation. (i) The MVF device design consists of two inlets conjoining at the annular junction, a conical mixing region, and an outlet. (ii) Magnified view on the annular junction. Mixing is improved through vortex focusing. Reproduced from ref. with permission from Springer Nature, copyright [2022]. d| Schematic representation of the microfluidic devices for a two-stage formation of cationic liposome at the 1st MHF region and pDNA loaded lipoplexes at the 2nd MHF region. Reproduced from ref. with permission from Elsevier, copyright [2017]. e| The assembly (i) and structure (ii) of mNALPs in a microfluidic T-junction chip. Mixing of lipid solution and DI water at the nanolitre scale in microfluidic channels leads to rapid changes in solvent properties that drive particle formation. Reproduced from ref. with permission from the Royal Society of Chemistry, copyright [2017].
Fig. 5
Fig. 5. Micromixers based on chaotic advection. a| The schematic of lipid nanoparticle (LNP) small interfering RNA (siRNA) formulation strategy employing the staggered herringbone micromixer (SHM). Lipids in ethanol and siRNA in aqueous solution are pumped into the two inlets of the microfluidic device to produce lipid nanoparticles. Reproduced from ref. with permission from the American Society of Gene & Cell Therapy, copyright [2012]. b| The schematic diagram for the design of a parallelized microfluidic device containing 4 rows of 32 mixing channels (i), highlighting the individual mixing unit design with a top view and a side view (ii) and the individual mixing cycle design with a top, angled, and side view (iii). The direction of flow is indicated by white arrows. Schematics are not to scale. Reproduced from ref. with permission from the American Chemical Society, copyright [2021]. c| Three-dimensional and top views of the iLiNP device with the basic structure of 20 baffle mixer structure sets. Reproduced from ref. with permission from the American Chemical Society, copyright [2018].
Fig. 6
Fig. 6. Micromixers based on Dean flows. a| Geometry and 3D model of a periodic disturbance micromixer (PDM). 90 semicircular structures were fabricated in the chip to generate Dean flows for mixing lipids in ethanol and water. Reproduced from ref. with permission from the American Chemical Society, copyright [2021]. b| Lipid/polymer hybrid nanoparticle production using the toroidal micromixer (TrM). Reproduced from ref. with permission from Elsevier, copyright [2022]. c| Nanoprecipitation of lipid-polymeric NPs in an MHF-SAR integrated device. Reproduced from ref. with permission from the American Chemical Society, copyright [2010]. d| Applications of lateral structure to laminar, serpentine zig-zag and split and recombine micromixers, respectively. Reproduced from ref. with permission from Elsevier, copyright [2020].
Fig. 7
Fig. 7. Microfluidic refinements for hydration. a| Schematic representations of the design of Y. Lin et al. Two 4 mm diameter wells were formed by bonding 2 mm thick PDMS to glass. The two cavities were connected by a channel. One cavity was for lipid film accommodation and hydration buffer injection to produce liposomes, and the other was for pumping out buffer. Reproduced from ref. with permission from Elsevier, copyright [2006]. b| Schematic drawing of the micro-tube system designed by H. Suzuki et al. Lipid chloroform solution was first injected to the 50 mm position of the microtubes with the same total length of 1.5 mm and various diameters of 200, 320 and 530 μm. After the lipid film formed by desiccator drying, PBS was pumped in and washed the microtubes, and the effluent was collected. Reproduced from ref. with permission from the Society of Chemical Engineers, Japan, copyright [2008]. c| Schematic illustration of K. Kitazoe et al.'s touch-and-go lipid wrapping technique. This technique constructed multifunctional envelope-type gene delivery nanodevices (MENDs) in two steps: (i) lipid coating in the microfluidic device and (ii) MEND formation in the microfluidic device. The top panel illustrates the mechanism of MEND formation based on the electrostatic interaction: the positively charged condensed plasmid DNA touched the lipid films on the glass, the substrate was wrapped in the lipid bilayer, and released as the MENDs. Reproduced from ref. with permission from the Royal Society of Chemistry, copyright [2011].
Fig. 8
Fig. 8. Schematic representation of cell-sized lipid vesicles. To simulate cells or function as bioreactors, ideal platforms require good encapsulation of biochemical materials, higher-order compartmentalisation, extracellular and intracellular communication, and replication of cellular metabolism.
Fig. 9
Fig. 9. Microfluidic refined phase transfer. a| Mechanism of bulk emulsion phase transfer. W/O emulsion is first generated by mixing the lipid oil phase and the inner aqueous solution (usually sucrose buffer). Then the emulsion is transferred onto the top of the outer aqueous solution (usually glucose buffer). After centrifugation, the oil phase is removed, and the pellet is resuspended to yield GUVs. b| Schematic of vesicle preparation through microfluidic emulsification and bulk template transfer. The aqueous phase containing the target encapsulated species is first emulsified in lipid-dissolved oleic acid for stable lipid emulsions and then injected into an aqueous mixture consisting of ethanol and water to remove the oleic acid. Reproduced from ref. with permission from the American Chemical Society, copyright [2006]. c| A microfluidic device for generating GUVs or LUVs in two steps. (i) Schematic of the different layers used to create the final microfluidic device. An aqueous solution containing molecules to encapsulate is pumped into the first input channel (blue). Oil solvents saturated with lipids are pumped into the second input channel (yellow). These two channels are separated by a layer of polycarbonate filter. Droplets are formed by driving the aqueous solution through the rigid filter into the oil phase under cross-flow emulsification conditions. (ii) An image of a single microfluidic device. The outlet channel has been outlined to help with visualization. (iii) Emulsion phase transfer of lipid-stabilized microscale or nanoscale droplets through a lipid-rich interface to form GUVs or LUVs. Reproduced from ref. with permission from John Wiley and Sons, copyright [2019].
Fig. 10
Fig. 10. Microfluidic single emulsion transfer. a| Schematic of microfluidic droplet transfer assisted by a triangle post. The triangular post skimmed the oil flow and deflected the preformed W/O droplets along its hypotenuse into the extracellular aqueous phase (AQex). As droplets traverse the interface, a second lipid monolayer is coated and GUVs are formed (micrograph scale bar = 100 μm). Reproduced from ref. with permission from the American Chemical Society, copyright [2011]. b| 2D schematic of microfluidic droplet transfer assisted by micro-step. The W/O droplets were transformed from the oil channel into a wider and deeper aqueous channel, where they picked up a second lipid monolayer from small vesicles in the Aqex. Reproduced from ref. and with permission from the Royal Society of Chemistry, copyright [2015 and 2016]. c| Schematic of microfluidic droplet transfer assisted by hydrodynamic traps (left). W/O droplets were generated by focusing flow, travelled through the delay line, and trapped by an array of cups. Schematic of layer-by-layer assembly (right (i)–(viii)) new phase boundaries were successively driven over the trapped droplets, and new lipid monolayers were deposited (micrograph scale bar = 100 μm). Reproduced from ref. with permission from Springer Nature, copyright [2013]. d| Formation and analysis of droplet-stabilized GUVs. The copolymer-stabilized W/O droplets (dsGUVs) were separated at a T junction by a tributary oil flow containing 20 vol% destabilizing surfactants. The passive trapping structures drained the oil phase into adjacent outlets, and GUVs were released as the droplets entered the aqueous phase. Scale bars, 20 μm. Reproduced from ref. with permission from Springer Nature, copyright [2017].
Fig. 11
Fig. 11. Microfluidic double emulsion-based vesicle generation. a| (i) Top: Formation of phospholipid-stabilized W/O/W double emulsion in a glass microcapillary device. Bottom: Optical micrograph of the double emulsion collected. (ii) Top: Vesicle formation through solvent drying on the vesicle surface. Excess phospholipid is concentrated in the remaining oil drop attached to the resulting vesicle. Bottom: Release of a vesicle from a double emulsion drop pinned on a glass slide. The oil drop that contains excess phospholipids remains on the glass slide. Reproduced from ref. with permission from the American Chemical Society, copyright [2008]. b| Fabrication of liposomes with distinct multicompartments. Schematic (top) and snapshots (bottom) of the fabrication of double emulsions with two distinct droplets. Scale bars are 100 μm. Reproduced from ref. with permission from the American Chemical Society, copyright [2016]. c| Top: Schematics of the microfluidic preparation of double emulsions with distinct interior liposomes (liposomes-in-liposome) and the dewetting process. Bottom: The formation of triple vesosomes (liposome-in-liposome-in-liposome) and the resultant structures. Scale bars, 100 μm. Reproduced from ref. with permission from the American Chemical Society, copyright [2017]. d| Schematics showing octanol-assisted liposome assembly (OLA) vesicle production and purification. An overall layout of the microfluidic device and the post-junction channel (left). A top view (right top) and a side view (right bottom) of the OLA junction. IA, inner aqueous phase; LO, lipid-carrying organic phase; OA, outer aqueous phase. Reproduced from ref. with permission from Springer Nature, copyright [2016].
Fig. 12
Fig. 12. Continuous droplet interface cross encapsulation (cDICE). a| (i) Schematic side view and working conditions of the cDICE setup. Abbreviations and physical variables are explained in the body text. (ii) Examples of the suspensions encapsulated in the vesicles. From left to right: 1-micron polystyrene colloids at 4% v/v, red blood cell, thin and thick actin filament bundles with fascin. The scale bar is 10 mm in all panels. Reproduced from ref. with permission from the Royal Society of Chemistry, copyright [2011]. b| Formation of GUVs by the droplet shooting and size-filtration (DSSF) method. (i) Capillary-based microfluidic device. (ii) Generation of GUVs and mechanism of size-filtration (within the rectangle shown in (i)). (iii) Two-step preparation of asymmetric GUVs in DSSF. Reproduced from ref. with permission from John Wiley and Sons, copyright [2015].
Fig. 13
Fig. 13. Pulsed jetting. a| (i) Conceptual diagram of the pulsed jetting method. The green area represents organic solvent. (ii) Sequential images of vesicle formation captured by a high-speed CCD camera. Reproduced from ref. with permission from the American Chemical Society, copyright [2007]. b| Illustration of a mimic exocytosis system of cell-sized lipid vesicle containing small vesicles using a triple-well device. Reproduced from ref. with permission from the Royal Society of Chemistry, copyright [2019]. c| Left: Two-droplet chamber configuration. SUVs delivered GFP-Cldn4 onto the lipid membrane by fusion. Right: Pulsed jetting based on lipid membrane with GFP-Cldn4 on it. Reproduced from ref. with permission from Biologists, copyright [2019]. d| Schematic images of sequential asymmetric GV generation with various lipid combinations. Various asymmetric GVs could be fabricated by aligning the single outer well to inter wells containing different lipid compositions and the conducting pulsed jetting. Reproduced from ref. with permission from Elsevier, copyright [2018].
Fig. 14
Fig. 14. On-chip electroformation. a| Mechanism of conventional electroformation. Lipid film is coated on the surface of the electrode, usually indium tin oxide (ITO) slides. An electric field is applied across the lipid film and surrounding buffer. The lipids interact with the aqueous solution and electric field by “peeling off” the electrode surface in layers and self-assembling into vesicles. b| Schematic of electroformation in a microfluidic device developed by Kuribayashi et al. The glass slides were coated with ITO electrodes and clamped a silicone sheet containing microfluidic channels where the electroformation occurred. Reproduced from ref. with permission from IOP Publishing, copyright [2006]. c| (i) Schematic diagram of on-chip giant vesicles electroformation process developed by Wang et al. (ii) Protruding microelectrode array with spatially non-uniform electric field. And (iii) planar electrode array with uniform electric field. Reproduced from ref. with permission from Elsevier, copyright [2013]. d| (i) Exploded 3D diagram of microfluidic electroformation device developed by Paterson et al., showing 1,1′ clamps; 2 lipid-coated ITO-coated slide; 3 PDMS sheet and 4 ITO-coated slide, arranged into a glass–PDMS–glass sandwich. (ii) Plan view of chip design (top), showing the electroformation and microtrap analysis chambers, connected by microfluidic channels (1), also depicted are the (2) wash and (3) peptide channels, as well as a collective outlet for waste (4). (iii) The microtrap array region was fabricated to capture GUVs for imaging analysis, of which the SEM image (bottom left, scale bar represents 50 μm) and fluorescent image of GUVs within it (bottom right, scale bar represents 50 μm) are presented. Reproduced from ref. with permission from the Royal Society of Chemistry, copyright [2014].
None
Yu Cheng
None
Callum Hay
None
Suchaya M. Mahuttanatan
None
James Hindley
None
Oscar Ces
None
Yuval Elani
See this image and copyright information in PMC

References

    1. Ryman B. E. Tyrrell D. A. Front. Biol. 1979;48:549–574. - PubMed
    1. Walde P., Preparation of vesicles (liposomes), in Encyclopedia of Nanoscience and Nanotechnology, ed. H. S. Nalwa, American Scientific Publishers, 2004, vol. 9, pp. 43–79
    1. Martin S. and Berg M., Biology, 10th edn, 2015, p. 107
    1. Kumar V. Complementary molecular shapes and additivity of the packing parameter of lipids. Proc. Natl. Acad. Sci. U. S. A. 1991;88:444–448. - PMC - PubMed
    1. Bangham A. D. Horne R. W. J. Mol. Biol. 1964;8:660–668. - PubMed