Giant Unilamellar Vesicles (GUVs) Production using Microfluidics

Traditional GUV Production Methods

While methods for producing nanometer-sized liposomes are well established in the field of healthcare as drug vectors, their use as engineered cells has been hampered by the lack of suitable methods for forming micrometer-sized vesicles.

Currently, the most common bulk methods for producing GUVs include gentle hydration, gel-assisted formation, and electroforming.^5,6,7 However, these methods are slow, unreliable, and offer little control over the unilamellarity, size, and monodispersity of the vesicles. In addition, they do not allow high, uniform encapsulation of large and charged biomolecules. Another bulk method, known as the droplet transfer method, overcomes some of these drawbacks, while still presenting a severe limitation in terms of size control. 

A. Gentle Hydration

In the conventional approach to forming Giant Unilamellar Vesicles, lipid films are created by dissolving lipids in chloroform at a concentration of approximately 1 mM inside a glass container.  

The films are then dried with argon gas. Subsequent hydration using pure water or a buffer solution results in GUV formation after an incubation period at 25°C, typically lasting from a few hours to several days.  

However, this method has drawbacks, including its slow pace due to extended incubation times, inefficiency resulting from poorly controlled lipid film swelling, and a broad size distribution of vesicles (ranging from a few micrometers to over 100 μm) with a lack of unilamellarity. Additionally, the formation of microsized unilamellar vesicles is limited to low ionic concentrations (up to 10 mM NaCl)^{3, 8}, 9). 

To address these challenges and enhance the approach, incorporating glass beads as a solid support for lipid coating can significantly improve the surface area for efficient lipid swelling and vesicle formation. This modification has been applied to various lipids, including both single-component lipids and mixed lipid systems.  

While the method demonstrates good encapsulation efficiency under physiological lipid and buffer conditions, resulting vesicles remain polydisperse, and there is a lack of control over leaflet asymmetry (referring to the difference between the lipid composition and/or physical properties of the inner and outer layers of the lipid bilayer)^{10, 11}.

B. Electroformation

The Electroformation of Giant Unilamellar Vesicles method offers a solution to the sluggish swelling times observed in gentle hydration techniques. In this process, an alternating electric field is applied to a dry lipid film within a formation chamber containing two platinum electrodes, inducing the formation of surface-attached vesicles through hydration.  

Alternatively, the lipid film can be deposited on glass slides coated with a conductive material, such as indium tin oxide, and separated by a spacer. This modification enables parallelization and high-throughput production of giant vesicles, making it a cost-effective approach.

Notably, the addition of an electrolyte in water, as opposed to water alone, can further expedite the electroformation process. However, it is crucial to avoid excessive concentrations of charged lipids in the mixture, as they may interfere with GUV production. 

Despite its advantages, including the preparation of unilamellar vesicles using physiological lipids and buffers within specific parameters, electroformation faces challenges in controlling leaflet asymmetry and achieving optimal encapsulation efficiency^{10, 12, 13, 14}. 

C. Gel-assisted hydration

Gel-assisted hydration allows the production of GUVs at physiological ionic strength. A hydrogel-forming polymer is dried to deposit a lipid film on a glass surface. Then, the film and the hydrogel are hydrated with a buffer solution so that giant unilamellar vesicles are formed at the gel/water interface. The size of the vesicles produced could be raised by increasing the buffer ionic strength.  

This method is faster than gentle hydration and electroformation and it can be used at physiological ionic strength as well as for different lipid compositions. Also, the risk of degrading the lipids faced by electroformation does not arise. By employing the gel-assisted hydration method, it is possible to create GUVs with specific lipid composition and buffer, allowing a degree of control over size and biomolecule encapsulation.  

D. Droplet transfer method

The droplet transfer method consists of introducing an aqueous buffer solution (forming the inner solution of giant vesicles) into an organic solution containing lipids. A water-in-oil (w/o) emulsion is formed by pipetting or vortexing. The emulsion is then placed in a microtube containing an aqueous buffer solution (forming the outer phase of the vesicles) and the lipids solution which will form the second lipid bilayer.  

These two phases are immiscible and thus form two heterogeneous layers. By gravity, the w/o emulsions are transferred to the interface between the lipid and buffer solutions, thus forming the outer solution of the giant vesicles.

Consequently, the giant vesicles with the lipid bilayer are formed from the w/o emulsions. Compared to the gentle hydration method, this technique gives more monodispersed, unilamellar, and well-encapsulated giant vesicles but with oil remnants in the membrane, which can alter properties like viscosity. Also, this process allows only low incorporation of cholesterol due to its hydrophobic nature. The formation and stability of droplets plays a crucial role in the creation of giant unilamellar vesicles using this approach.  

Therefore, controlling the size of these droplets allows for GUV size regulation, which is not attainable through the bulk water-in-oil (w/o) methods (pipetting/vortexing).^17,18,19

How does microfluidics enhance the GUV production process?

Microfluidics, through water-in-oil-in-water (W/O/W) double emulsions with lipids in the oil phase, emerged as a promising tool for GUV production compared to the traditional bulk methods and their drawbacks.   

Typically, the production of giant unilamellar vesicles in microfluidics is a two-step process, as depicted in figure 6. 

In the first step, an aqueous solution is pinched by an organic phase containing dissolved lipids. This results in the formation of water-in-oil (W/O) single emulsion droplets with a monolayer of lipids assembled at the interface. The single emulsion is then sheared into droplets by a second aqueous solution to form W/O/W double emulsions. Lipids can be present in the outer aqueous solution in the form of small vesicles and will form the second layer at the interface. The w/o/w method produces vesicles at high throughput, demonstrating effective size control and high encapsulation efficiency.  

However, the presence of residual oil in the bilayer is a recurring problem when using these processes for vesicle production.²⁰ 

Another option is to use a partially water-miscible oil, such as octanol, as the shell phase.⁸When octanol-containing lipids are extracted in water, lipids assemble along both interfaces to form giant unilamellar vesicles (figure 7).

This technique, first described by Deshpande et al. (2016), consists of forming double emulsion droplets from an aqueous core phase and an ultra-thin organic shell phase (lipid dissolved in 1-octanol) flowing through the outer aqueous phase (continuous phase). The droplets formed present an octanol pocket on one side of the droplet, in the direction of the flow. After a few minutes, oil-free vesicles are obtained after full detachment of the octanol pocket due to the dewetting. Next, the created vesicles and octanol droplets can be separated thanks to their density difference. 

This microfluidic technique allows efficient encapsulation of biomolecules with precise size control of the vesicles. In addition, it presents the opportunity to automate the process and create a more complex arrangement of leaflets while enhancing control over the number and composition of each leaflet.  

However, a question remains regarding the biomimetic properties of the lipid vesicles due to the usage of surfactants and additives in both the aqueous phases that can affect the biophysical membrane properties, hampering their use as cell mimics.^9,10 

Yandrapalli et al. succeeded in generating giant unilamellar vesicles without the use of additives or surfactants by using a well-designed PDMS-on-glass chip with a double flow focusing junction.²¹ This design enabled the generation of vesicles in various sizes, ranging from approximately 10 to 130 μm, using either neutral or charged lipids and under physiological buffer conditions. 

Characterization tests confirmed the purity, functionality, and stability of these vesicle membranes through lipid diffusion, protein incorporation, and leakage assays.

Furthermore, their potential as artificial cells was demonstrated by increasing their complexity, such as encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres.^10,21

Overall, the use of microfluidics to produce microsized unilamellar vesicles overcomes most of the limitations encountered in batch processes. It allows a high level of control over size and unilamellarity, as well as a high production rate and high encapsulation efficiency.

GUV Applications

The applications of giant vesicles can be categorized into two main areas: understanding membrane biophysical processes and the construction of synthetic cells.  

In the field of membrane biophysics, giant vesicles have been used to study phenomena such as membrane phase separation, fluid/gel-like domains, and the effects of proteins and specific lipids on membrane properties. These studies have led to insights into various biological processes, including fission, fusion, and shape changes in cells.

Additionally, researchers have explored the interaction of antimicrobial peptides and the transport properties of membrane proteins in giant vesicles.^10,22,23,24

In the context of building synthetic cells, giant vesicles have served as an initial component. They have been used to mimic cellular features such as compartmentalization, communication, motility, growth, and reproduction. This includes the creation of vesicle-in-vesicle structures resembling cell organelles and the development of systems that enable enzymatic reactions and regulated transcription.

Controlled growth, budding, and division of giant vesicles have been achieved through various external and internal stimuli.

Furthermore, synthetic cells constructed from giant vesicles have been engineered to communicate with natural cells and to perform functions such as replication of genetic material, self-reproduction, and self-organizing protein expression.^10,25,26

In summary, creating GUVs for artificial cells involves two key goals: developing a complex and biologically analogous membrane while achieving a multicomponent lumen. These requirements ensure that GUVs accurately mimic the properties and functions of natural cells, making them suitable containers for artificial cells. Therefore, the fabrication method of giant unilamellar vesicles remains crucial.²⁷   

Giant vesicles have also found applications in drug delivery, including non-intravenous routes. The development of microfluidic-based high-throughput techniques has expanded the possibilities for encapsulating larger therapeutic molecules like DNA, RNA, and enzymes, making them suitable for engineering complex cargo delivery systems responsive to external triggers.^28,29