Lipid Nanoparticles / Liposome Synthesis
by NanoGenerator™ Microfluidic Mixing System
Lipid nanoparticles (LNPs) or solid lipid nanoparticles (SLNs, sLNPs) are nanoparticles composed of lipids and possess a solid lipid core matrix which is able to solubilize lipophilic molecules. Typically they are spherical with an average diameter between 10 and 1000 nanometers.
Lipid nanoparticles have emerged across the pharmaceutical industry as promising vehicles to deliver a variety of therapeutics. They are a novel pharmaceutical drug delivery system and formulation. LNPs became more widely known in late 2020, as some COVID-19 vaccines that use RNA vaccine technology coat the fragile mRNA strands with PEGylated lipid nanoparticles as their delivery vehicle.
Liposomes, an early version of LNPs, are a versatile nanomedicine delivery platform. A number of liposomal drugs have been approved and applied to medical practice. A liposome is a spherical vesicle with at least one lipid bilayer. The liposome can be used as a vehicle for administration of nutrients and pharmaceutical drugs. Liposomes are composite structures, consisting of phospholipids, especially phosphatidylcholine. It may contain small amounts of other molecules. Though liposomes can vary in size from low micrometer range to tens of micrometers, unilamellar liposomes, shown as the Figure below, are typically in the lower size range with various targeting ligands. The ligands are attached to their surface allowing for their surface-attachment and accumulation in pathological areas for treatment of disease.
Due to their particular properties such as biocompatibility and biodegradability, LNP and liposome have been becoming an important nanostructure in the field of encapsulation and delivery for bioactive agents. A variety of bioactive materials could be incorporated into LNPs including cosmetics, food ingredients, and pharmaceuticals. Meanwhile, accompanied by their nanosize they have potential applications in nanomedicine, cosmetics, and food industry.
Liposome composite structure 
The microfluidic technology based miniaturized reactors enable the rapid mixing of reagents, the control of temperature, and the precise spatial-temporal manipulation of reactions. The controlled and homogeneous mixing in microfluidic synthesis methods results in smaller and uniform nanoparticles. The physicochemical properties of nanoparticles can be precisely controlled in a reproducible manner. The control of the reaction environment leads to improve the quality of nanoparticle size distribution, better size reproducibility, and eventually improve the preparation process yield of nanoparticles.
Different nanoparticle synthesis, such as semiconductor nanoparticles, metal nanoparticles, colloidal nanoparticles, and biomaterial nanoparticles, have been demonstrated in microfluidic devices in homogeneous and well controlled fashion . Precigenome PG-MFC controller, as a precise pressure control instrument, is very suitable for these nanoparticle synthesis applications.
PreciGenome NanoGenerator Flex™ System consists of high-precision flow controller and microfluidic mixing cartridge as shown in the Figure below. Since flow rates in these experiments are large enough (milliliters per minute range), they can be estimated by weighing collected nanoparticle solutions within certain time period. Alternatively, liquid flow sensors can be added to flow line of reagents to monitor the flow rates.
PreciGeome Liposome Nanoparticles Synthesis System Setup
Microfluidic mixer chip
Microfluidic chips used in the experiments are a microfluidic mixing chip (passive mixing, herringbone mixing). Solution A and Solution B are loaded in 50mL reservoir kits (PG-MRK-50ML). In liposome synthesis experiments, Solution A is lipid in IPA. Solution B is water. During experiments, preset pressures from PG-MFC pressure controller were applied to reservoir kits. Solutions in reservoir kits were pushed through tubings into the two inlets of a microfluidic chip and mixed inside the channel of the microfluidic chip. The mixed solution (nanoparticle solution) was collected from the outlet of the microfluidic chip. Users can optimize the mixing ratio, flow rates, and synthesis effect by changing the pressure settings using the pressure controller.
In general, lipid mixture is dissolved in solvent phase, such as IPA. This forms so called “Oil” (solution A). DI water is used as “water” phase (solution B). DLS is used to measure particle average size and PDI. We measured the liposome particle size with different total flow rates when we fixed the flow ratio of water to oil phase as shown in the Figure below. There is a trend that the larger the total flow rates, the smaller particle size we can get. The particle size ranges between 80nm to 400nm.
Figure. total flow rate vs liposome size
We also investigated the relationship of liposome size and water/oil phase pressure/flow ratio. When the W/O pressure/flow ratio increases, the average liposome particle size decreases as shown in the Figure below.
Figure. W/O pressure/flow ratio effect on liposome particle size
Similar to PLGA nanoparticle synthesis, the relationship between nanoparticle PDI and flow rates and flow ratios is not conclusive. The PDI of liposome nanoparticles ranges from 0.25-0.8. Further investigation is needed. For example, we can compare PDI with different mixing methods (diffusion based mixing vs. Herringbone mixing).
In this study, we further studied DNA encapsulation efficiency by lipid nanoparticles and DNA encapsulation effect on particle sizes. We observed that the lipid nanoparticle size increases with DNA encapsulation under the same pressure conditions as shown in the Figure below. By tuning the formulation of lipid mixture in IPA, we obtained more than 95% DNA encapsulation efficiency. The Table below summarizes the encapsulation efficiency.
Figure. Liposome size with or without DNA encapsulation at different flow rates
Torchilin, V (2006). "Multifunctional nanocarriers". Advanced Drug Delivery Reviews. 58 (14): 1532-55.
Chiesa, E, et.al., The Microfluidic Technique and the Manufacturing of Polysacharide Nanoparticles, Pharmaceutics, 2018, 10:267-289