Lipid nanoparticles (LNPs) are spherical lipid vesicles for nucleic acid delivery. LNPs are generated spontaneously when an ionizable lipid and a nucleic acid are mixed. However, the traditional pipetting methods can result in heterogeneous and larger particles that fail to meet encapsulation efficiencies.
The relative amounts of ionizable lipid, helper lipid, cholesterol, and PEG significantly influence the functionality of a lipid nanoparticle. Formulation parameters need to be optimized to improve the performance of a lipid nanoparticle.
What is Lipid Nanoparticle Formulation?
Lipid nanoparticles (LNP) are nano-sized particles mainly composed of lipids. They are often referred to as liposomes, microemulsions, or nano-emulsions. They can be formulated into various structures, such as spherical vesicles, multi-lamellar vesicles, elliptical vesicles, microspheres, and other nanostructured vesicles.
The lipid composition of LNP can significantly affect their stability, biodistribution, and delivery efficacy. For example, cholesterol can enhance particle stability, and the presence of cholesterol derivatives in the formulation can also promote the formation of less rigid and more permeable bilayers. The molecular geometry of cholesterol can further influence the cellular uptake of lipid nanoparticles and improve their in vivo delivery to specific organs. For instance, phosphatidylcholine-mRNA formulations containing cholesterol analogs with shorter alkyl phytosterol chains show higher accumulation in liver endothelial cells than in hepatocytes123.
In addition to lipids, LNP formulations typically contain other components that can further optimize the particle properties.
Lipid Nanoparticle Formulation Techniques
There are many techniques for lipid nanoparticle formulation, including liposomes, emulsions, nitric oxide-based nanoparticles, solid lipid nanoparticles (SLN), and nitric oxide-encapsulated RNA (NLPR). Each has its advantages and disadvantages. For example, the liposome preparation involves dissolving lipids in an organic solvent and slowly injecting the mixture into an aqueous phase containing surfactant.
Liposomes with a high drug loading and lipid-to-water ratio can be made using this simple-to-use approach. It can, however, yield an increased number of heterogeneous particles with diameters ranging from 70 to 200 nm and necessitates the use of hazardous chemical solvents.
Other methods include cold homogenization and hot homogenization. Cold homogenization uses a high-speed stirrer to create a pre-emulsion of the active ingredient and the lipid matrix at a temperature lower than their melting points. This is followed by rapid stirring and sonication to form an emulsion containing a particle size of several microns. After creating an emulsion, the lipids are rapidly cooled to obtain the final lipid nanoparticles.
This method can encapsulate various compounds, including hydrophobic drugs and ionizable molecules. Lipid-based NLPRs are an up-and-coming platform for the delivery of RNA because they can evade endosomal degradation and deliver siRNA to target cells.
How to Formulate Lipid Nanoparticles
Lipid nanoparticles (LNPs) are lipid vesicles that encapsulate payloads such as nucleic acids. LNPs can be classified into several types, including liposomes, solid lipid nanoparticles, nanostructured lipid carriers, and microemulsions.
The lipid composition of an LNP depends on the desired physicochemical properties. For example, the molar ratio of an ionizable cationic lipid to a non-ionizable or helper lipid influences the particle size and zeta potential. Moreover, the lipids used in an LNP can impact its biological behavior.
For example, the lipids in the mRNA-LNP formulations for the two approved COVID-19 vaccines developed have specific characteristics that lead to effective cellular uptake. These lipids have one or more tertiary amines that can be protonated in the acidic environment of the endosome, which disrupts the membrane and releases the mRNA into the cell.
The lipids also have long alkyl tails, which help them form a cone-shaped structure. This is thought to help the mRNA escape from endosomal degradation and into the cytoplasm. Additionally, the lipids are coated with polyethylene glycol, improving the particles’ stability and allowing for more efficient cellular uptake. Finally, the lipids are designed to be stable against shear, which is important to achieve a uniform distribution of mRNA.
Lipid Nanoparticle Formulation Parameters
Lipid nanoparticles are currently the clinic’s most advanced non-viral gene delivery system. They deliver siRNA or mRNA into cells to silence or modify target genes and can also activate immune cell responses. Combined with a checkpoint inhibitor, they are shown to improve patient outcomes in head and neck squamous cell carcinoma.
The lipids used in LNPs contain ionizable cationic lipids (DLin-MC3-DMA) and neutral phospholipid (1,2-distearoylsn-glycero-3-phosphocholine [DSPC], cholesterol or polyethylene glycol-modified lipids. The ionizable lipid initiates particle formation and facilitates membrane fusion during the internalization of the nucleic acid cargo. Once inside the cell, DLin-MC3-DMA is replaced by Apolipoprotein E (ApoE), which delivers the mRNA into the cytoplasm.
Zwitterionic ionizable lipids can be used to modulate the cellular response. The mRNA is then taken into the nucleus, which can be translated into protein in vivo or edited by RNA-directed methylation (RDM) to achieve desired therapeutic functions. Developing lipid nanoparticles with desired properties requires accurate, high-throughput, low-volume measurements of subvisible particles’ physical and chemical stability. Backgrounded membrane imaging, a contemporary form of microscopy, offers the accuracy and speed required to measure subvisible particles in a clinically relevant test system from early-stage inception to product release.
