Lipid nanoparticles (LNPs) are a type of nanocarrier that encapsulate therapeutic agents (nucleic acids, small molecules, etc.) within lipid-based structures. They've gained enormous attention as effective drug delivery vehicles, especially since their successful use in both the Moderna and Pfizer SARS-CoV-2 vaccines. Each vaccine deployed LNPs to deliver messenger RNA (mRNA) to the cytosol of target cells without damage. However, lipid nanoparticle formulation isn't limited to intravenous therapeutics. Research into LNPs for drug delivery has explored various administration routes, including oral, topical, and pulmonary options. Yet, despite the burgeoning success, greater development of lipid nanoparticle formulation remains hindered by aggregation issues.
What are Lipid Nanoparticles?
LNPs are spherical assemblies of lipid and/or lipid-like molecules, including phospholipids and ionizable lipids. The former comprises the basic structural framework that encapsulates the drug, while the latter aids in drug release due to its pH-dependent charge. Lipid nanoparticle formulations remain attractive for a number of therapeutics, given their enhanced bioavailability, stability, and ability to target specific sites within the body. Various formulation methods exist, but common approaches include nanoprecipitation, microfluidics, and high-pressure homogenization, which enable precise control over particle size, encapsulation efficiency, and stability.
However, like protein-based biotherapeutics, LNPs are prone to aggregation when exposed to stressors like freeze-thaw cycles or agitation. This can reduce stability and increase particle size, which could impair targeting and decrease bioavailability, leading to a host of complications. Let's explore some of the factors influencing LNP aggregation.
Factors Influencing LNP Aggregation
Particle Size and Surface Properties
The size of nanoparticles plays a crucial role in their aggregation behavior. Smaller nanoparticles tend to have a higher surface energy, which can lead to aggregation as they seek to minimize this energy by clustering together. Additionally, the surface charge and hydrophobicity of the nanoparticles influence their tendency to aggregate. For instance, hydrophobic interactions can drive aggregation in lipid environments.
Lipid Composition
The types of lipids used in LNP formulations affect their stability and propensity to aggregate. Charged lipids, particularly cationic lipids, can interact with plasma proteins like fibrinogen, leading to aggregation and potential clotting issues. Helper lipids such as phospholipids and cholesterol are crucial for maintaining structural integrity and preventing aggregation by stabilizing the lipid bilayer.
Environmental Conditions
Changes in temperature, pH, and mechanical stress can impact the stability of LNPs. For example, freeze-thaw cycles can cause LNPs to aggregate by disrupting the lipid bilayer structure, leading to increased subvisible particle formation. Similarly, heat stress can influence the solubility and aggregation behavior of LNPs.
Interactions with Biological Membranes
When LNPs interact with biological membranes, their aggregation is influenced by the membrane's properties, such as its bending modulus and the strength of interaction between nanoparticles and lipid headgroups. These interactions can lead to different wrapping regimes around the nanoparticles, affecting their aggregation patterns.
PEGylation
While PEGylated lipids are used to enhance circulation time and reduce clearance by the mononuclear phagocyte system (MPS), they also play a role in preventing aggregation by providing a steric barrier that reduces protein adsorption and cellular uptake. However, this "stealth effect" can also decrease transfection efficiency, presenting a trade-off in formulation design.
Exploring Lipid Nanoparticle Formulation Stability
We conducted a study demonstrating how the FlowCam 8100 and FlowCam Nano can be used to analyze aggregation and particle formation in LNP formulations. These formulations were exposed to accelerated stability conditions (freeze-thaw stress and heat stress) to induce aggregation and degradation. Using FlowCam 8100 and FlowCam Nano, we analyzed particle concentrations, sizes, and images of the stressed samples. The study results illustrate the combined value of FlowCam instruments in monitoring LNP biopharmaceutical formulations and guiding process improvement strategies.
Notably, while submicron particle sizes (0.3 µm - 1 µm) showed negligible differences between samples, the subvisible (> 1 µm) particle size distribution from heat-stressed samples was shifted toward larger sizes compared to particles generated by freeze-thaw stress. This indicates that heat stress had a greater negative impact on the LNP formulation.
The figure above includes images captured by FlowCam 8100 and FlowCam Nano, depicting particles from freeze-thaw- and heat-stressed samples. The LNP particles showed amorphous shapes similar to those of other API particles, such as protein aggregates. Some FlowCam Nano images highlighted in red boxed regions where particles appeared attached, potentially indicating dimeric LNP structures.
This finding suggests that FlowCam Nano can effectively monitor LNP aggregation even at early stages when only oligomeric aggregates are formed. For more information, download the application note to read the complete study.
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This observation suggests that FlowCam Nano can effectively monitor LNP aggregation even at early stages when only oligomeric LNP aggregates have been generated. Download the application note to read the full study.  |
