Liposome and Lipid Nanoparticle | An overview

This review gathers a collection of information regarding liposome and lipid nanoparticle from their use and applications to how to generate them by bulk – batch and microfluidic methods.

Research and industry have shown an increasing interest in liposome structures notably in the field of biological and/or biochemical applications. This trend led scientists to focus their efforts on developing specific uses of Liposomes for instance for drug delivery particles for targeted therapy.

This growing interest forced researchers to find innovative, reproducible, and well-controlled techniques for liposome generation and technologies. Indeed, many issues remained to be overcome related to batch preparation such as the batch dispersion, the particle average size, the encapsulation efficiency, and the poor payload management.

In this review, it is shown that microfluidics is a great candidate to answer many of those challenges, notably thanks to its well-controlled and scalable nature.

What are liposomes?

The term “Liposomes” describes a specific type of vesicle that is a supramolecular aggregate made of amphipathic molecules, commonly phospholipids, formed in an aqueous phase. [1] Liposomes are part of the family of lipid-based deliveries. The lipid-based delivery family comprises as well [1]:

• Single emulsion that uses surfactants to stabilize drops of oil in water or droplets of water in oil. 
• Micelles: particles composed of surfactants with their lipophilic tails forming the core.
• Nanoemulsions: particles with an oil core surrounded by a monolayer of surfactant.
• Solid-lipid nanoparticles or SLPs: particles with a solid lipid or wax core and monolayer or surfactants. 
• Nanostructured lipid carriers or NLCs: particles with a core blend of solid and liquid lipids, favored for resisting the crystallization SLPs are prone to that pushes contents out of the particle.

Liposomes are composed of one or more closed shells, or lamellae, made of a phospholipid bilayer and enclosing a small volume of aqueous liquid. Liposomes’ diameter ranges from tens of nanometres up to hundreds of micrometers depending on the production process and the application. [2]

Figure 1: Schematic representation of various lipid-based structures: (a) lipid molecule composed of a hydrophilic head and a hydrophobic tail (b) Self-assembled membrane structure made of lipid molecule, and (c) a lipid vesicle. [3]

Over the years, the reduction in size of the lipid-based delivery system (down to the 10- to 200-nm range) linked to the development of high-shear mixing methods, allowed the overall improvement of the particle suspension stability, shelf life and properties.  Typically, liposomes used in medical applications are unilamellar with an average size of ~100 nm.

What are the differences between liposome and other lipid nanoparticle?

The main difference compared to other lipid nanoparticles resides in the stabilization process at the interface: Instead unlike surfactant-stabilization, here phospholipids arranged into spherical cell membrane–like lipid bilayers with their water-loving “heads” toward the aqueous medium and their fatty “tails” tucked toward each other act as a stabilizing agent. [1] When Phospholipids molecules are dispersed in an aqueous solution, they spontaneously organize in liposomes to reduce the surface interaction between their hydrophobic chains and the surrounding water.  The behavior in the solution will vary on various parameters [1]: 

• Intrinsic parameters linked to the nature of the phospholipid molecule such as the chemical structure, the carbon backbone…
• Controlled parameters such as the concentration, the temperature, the mixing speed, and the injection flow rate. 

All these parameters will define the final liposomes dispersion properties. Among those properties, the size, lamellarity and size dispersion are key characteristics to control effectively depending on your application. As an example, typical liposomes used for medical applications are unilamellar (only one layer of lipids for the shell) and measure around 100 nm diameter but other structures exist as depicted in the figure below:

Figure 2 : Schematic representation of various vesicle size and lamellarity systems: Small unilamellar vesicles (SUV) are less than 100 nm in diameter; large unilamellar vesicles (LUV) are between 100 and 1000 nm; and giant unilamellar vesicles (GUV) are larger than 1 micron. Multilamellar vesicles have many membrane layers, and multivesicular vesicles encapsulate smaller vesicles. [3]

The structure (particle shape and shell nature) of liposomes allows researchers to trap certain molecules inside it. As an example, it is possible to trap a drug inside a liposome, and thus control its release in a specific area of the body. The next section of the review will introduce you to how liposomes and lipid nanoparticles can be employed for drug delivery applications.

Liposome technology: What are liposomes used for?

Liposomes or lipid nanoparticle have been used in many fields of applications in biology and biochemical industries:

• Cosmetics
• Food and Beverages Industry
• Drug and vaccine delivery

The next discussion will be dedicated to the use of liposomes for drug delivery.

Liposomes for drug delivery

SInce 1961, liposome and lipid nanoparticle  have been employed as delivery vehicles for transporting substances into the body by making mouth absorption easier. [1, 4, 5] or by preventing breakdown by stomach acid [6]. They have been considered as potential drug delivery systems because of their biocompatibility and ability to incorporate both hydrophilic and hydrophobic therapeutic agents. 

Liposomes as drug delivery systems present many benefits. One of the biggest advantages is the ability to  reduce the risk of toxic side effects, a significant issue for chemotherapeutics, which often exhibit high toxicity to healthy body organs and tissue [7-8] Recent studies illustrate the great potential for the widespread adoption of liposomes in cancer treatment. [9] In addition to their low toxicity, these structures have major characteristics including biocompatibility, lower clearance rates, the ability to target cancer tissues and controlled release of drugs. 

Depending on the size, lamellar number and form and formulation of constitutes, there are several types of liposomes. These systems have been used clinically for diagnostics, therapeutics and vaccine improvement. Drug and gene delivery are two therapeutic aspects, in which liposomes can be effective due to their specific properties. 

As an example, Liposome structure allows for the creation of pockets of entrapped water along with water soluble compounds and the protection from hostile digestive conditions and the ease of gastrointestinal uptake. Thus, the hydrophobic fatty acid core of the bilayers could simultaneously host hydrophobic compounds, inducing a small spherical package which could carry both hydrophilic and hydrophobic compounds. [10] Thus far, the main mechanism that has been found in the literature for the loading of drugs by liposomes is the encapsulation. The process of encapsulation will depend on the nature of the drug to be encapsulated. 

For the encapsulation of hydrophilic drugs, hydration of lipids hydrophilic drugs mixture is required. Thus, drugs can enter the liposome core and other materials remain in the outside part of the liposome. Remained materials will remove drug entrapment in the liposome. In order to purify these two parts (drugs and remaining outside materials), gel filtration column chromatography and dialysis are used. In addition, dehydration and rehydration methods may be applied for high encapsulation of the DNA and proteins.[11]

For the encapsulation of hydrophobic drugs, the phospholipid bilayer of liposomes is the region of focus. By encapsulating hydrophobic drugs, the movement of the latter is limited towards the outer aqueous and inner parts of liposomes. The encapsulation in the hydrophobic part of the liposome occurs by solubilizing the drug into an organic solvent with phospholipids. [12] 

In the last two decades, encapsulation has been performed by batch production methods. Despite the use of batch methods by industry for many years, it presented several drawbacks from the lack of control over the lipid nanoparticle size and distribution to the importance of waste of reagents in the reaction process. 

Those drawbacks led researchers to develop new methods to synthesize liposome or lipid nanoparticles such as microfluidics. Microfluidics is the science of handling fluids (liquid or gas) from the milli down to the nanometer scale. It is used for many applications from biological applications to droplet generation. 

As explained above, the behavior of the liposomes in solution, and therefore the controlled release in the case of drug delivery application will depend strongly on two kinds of parameters: the liposomes intrinsic properties and the controlled experimental parameters. 

Microfluidics as a tool to generate liposomes or as a technique used to tune the controlled release of the drug contained within a liposome presents many advantages to make your experiment a success. In the next section, the generation of liposomes by microfluidics will be discussed. In the following section, a microfluidic setup will be introduced, allowing for a high-throughput liposome and lipid nanoparticle synthesis. 

For more information about the use of microfluidics for drug delivery applications, please read the full review available here.

How to generate liposomes?

Synthesis by batch method

Figure 3: Schematic representation of the ethanol injection procedure. [2]

Batch or Bulk production methods have been commonly employed to prepare liposomes for many years. Bulk processes comprise [1]: 

• Vesicle extrusion through the pores of polycarbonate membranes 
• Treatment with ultrasound
• Repetitive freezing and thawing

Batch production methods are based on two processes:

• A rehydratation method where based on the swelling of initially dried pre-organised lipid films (i.e. rehydration methods), followed by the mechanical manipulation of the dispersed bilayers [1] 
• The second process implies the use of: (i) a cosolvent in which the lipids are soluble, (ii) an additional non-bilayer-forming “co-amphiphile”, or (iii) specific ionic species that influence the supramolecular aggregation of the lipids [1] 

These methods, even though being commonly used, show heterogeneous and poorly-controlled chemical and mechanical conditions during lipid vesicle formation, resulting in non-uniform vesicles in terms of both lamellarity and size poor reproducibility. This last point is critical when encapsulating expensive reagents such as pharmaceuticals or cosmetics and proved to be the limitation for a broader use of liposomes at large scale. Thus, additional post-processing steps are required to get homogeneous vesicle dispersions. 

In contrast, microfluidics presents tremendous advantages to overcome the issues encountered with liposome bulk preparation. Microfluidics being characterised by laminar flow conditions and diffusive mass transfer, allows for the production of liposomes with excellent control over size and lamellarity. In addition, unlike bulk methods, in situ monitoring of the liposome formation process, continuous production and scaling up by microreactor parallelisation becomes possible with microfluidics.[2]

Synthesis by microfluidics

Microfluidic methods to liposome nanoparticle synthesis comprise electroformation and hydration [13], extrusion [14], pulsed jetting [15], double emulsion templating [16], ice droplet hydration [17], transient membrane ejection [18], droplet emulsion transfer [19], hydrodynamic focusing [20] and herringbone micromixing [22]. 

Figure 4: Schematic representation of a microfluidic device employed for small unilamellar liposome synthesis. [2]

Liposome synthesis by herringbone micromixing

Microfluidic approaches to liposome nanoparticle synthesis are based on the application of various mixing devices in addition to the effect of shear forces, electric field or various microfluidic effects taking place in the microchannel. [21] Indeed, Lipid Nanoparticle Synthesis can be performed by microfluidics with a herringbone micromixer. This geometry allows a very fast and effective nanoprecipitation. The herringbone micromixing is based on microfluidic hydrodynamic flow focusing on an alcohol stream with two aqueous buffer streams. [22]

Figure 5: Schematic representation of the principle of microfluidic mixing in herringbone channel and formation of liposomes [21]

In this method, the chip presents a cross-flow geometry with a central flow and two side flows. The central flow contains an alcoholic solution (or any water-miscible solution) in which the phospholipids are dissolved. The side channels are adding water to the mix. 

Due to the low Reynolds number, the flows will merge in a very controlled manner either through diffusion of one fluid into the others (diffusion speed being controlled via the flow rate in the channels) or through induced mixing by using a specific micromixing chip such as a Herringbone mixer chip. Since the alcohol diffuses in water, the solubility of the phospholipids in the resulting solvent mixture is decreasing. When the solubility reaches its low threshold, the phospholipids spontaneously reorganize into liposomes.

Find below an example of microfluidic setup that can be used to perform liposomes and lipid nanoparticle synthesis:

Figure 6: Schematic representation of microfluidic setup to generate Lipid nanoparticles with a herringbone micromixer.

If you’re interested in reproducing this setup in your lab, check the full description of the setup here. 

This method, combined with pressure-driven flow controlled microfluidics, results in very reproducible results and highly monodispersity nanoparticles while using a limited amount of reagents. The resulting liposomes size ranges from 10 to 250  nm.

The production can be easily scaled up by including and parallelizing several herringbone micromixers instead of one, thus increasing the throughput of the system without sacrificing monodispersity and response time.

Lipid Nanoparticle Generation by double emulsion templating

Droplet microfluidics is commonly employed to generate single and double emulsions. Microfluidics for droplet production, unlike batch methods, allows for better control over parameters such as droplet shape, size, monodispersity, and frequency of generation. 

For those reasons, double emulsion templating, based on droplet microfluidics, was considered for lipid nanoparticle synthesis. 

To perform lipid nanoparticle production, a  double emulsion water-in-oil-in-water system needs to be generated. Interestingly, Tan et al. (2006) [23-24] proposed shear focusing microfluidic method to perform highly monodisperse 100 nm diameter droplet generation and subsequently microfluidic encapsulation of cells inside lipid vesicles without the use of any toxic lipid solvents. The encapsulation efficiency inside the vesicles was driven by the microfluidic flows. The bead encapsulated vesicle diameters ranged between 20 up to 60 μm. [24] In this work, the first step consisted in forming a water-in-oil (oleic acid) emulsion stabilized by lipids. The resulting emulsion was then transferred to ethanol. Doing so, the oleic acid phase dissolved in the ethanol and the phospholipids were forced to rearrange to form a bilayer membrane. [24]

With this process, microfluidics ensures an effective control over the whole experiment and the resulting experimental reproducibility to perform a successful experiment. The schematic diagram below shows how pressure-driven flow controlled microfluidics can be used to perform double emulsion encapsulation. For more information, feel free to contact our team of experts.

Figure 7: Schematic representation of microfluidic setup to perform lipid nanoparticle synthesis by double emulsion encapsulation

This microfluidic setup is based on the pressure-driven flow controlled microfluidic droplet pack by Elveflow. For more information, please check the application pack here. 

Unlike the hydrodynamic flow focusing method, the double encapsulation process allows to produce larger liposomes (> 1 µm) at high-throughput and with a comparatively smaller loss of reagents in terms of encapsulated chemicals.

Takeaway Note

This review presented an overview of liposomes and lipid nanoparticle synthesis including the principle, application and generation techniques (batch and microfluidics). Microfluidics was found to be a choice technique to perform lipid nanoparticle production due to its fine control over the experimental parameters defining the resulting liposomes nanoparticle properties. 

The following table sums up the three hereby-presented techniques and their characteristics.

Encapsulation efficiency = Encapsulated material / Waste material

References