Important Parameters of Lipid Nanoparticles (LNPs): Particle Size & Zeta Potential

Why Particle Size & Zeta Potential are Important Parameters for Lipid Nanoparticles?

Lipid nanoparticles are colloidal carriers composed of lipids and surfactants, holding vast potential for biological applications. They play pivotal roles in the delivery of bioactive molecules such as drugs, genes, vaccines, and nutrients. Types of lipid nanoparticles include solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), among others, with their properties and performance contingent upon factors like composition, structure, shape, size, and surface charge. Among these, particle size and zeta potential are two crucial parameters influencing the stability, biodistribution, cellular uptake, and therapeutic effects of lipid nanoparticles. Particle size determines the transport and permeation capabilities of nanoparticles in vivo, while zeta potential reflects their surface charge characteristics, impacting their interaction with the biological environment. Therefore, the modulation of particle size and zeta potential holds significant importance in optimizing the performance and applications of lipid nanoparticles.

Schematic diagram of LNP-mRNA.

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The Particle Size of Lipid Nanoparticles

What is the Particle Size of Lipid Nanoparticles?

Particle size refers to the diameter of spherical or granular particles with the same volume as the actual particles. Particle size influences the physical stability, solubility, dissolution rate, bioavailability, and targeting of lipid nanoparticles. Generally, smaller particles have a higher surface area-to-volume ratio, resulting in increased solubility and faster dissolution rates. Smaller particles also exhibit longer circulation times in the bloodstream and enhanced permeability and retention (EPR) effects in tumor tissues. However, smaller particles may also have a higher tendency to aggregate and lower loading capacity. The particle size of lipid nanoparticles can be influenced by various factors during their preparation process, such as the type and concentration of lipids, surfactants, drugs, solvents, temperature, pressure, stirring speed, homogenization methods, ultrasonication time, etc. Depending on the preparation method and conditions, the particle size of lipid nanoparticles can range from tens of nanometers to several hundred nanometers.

What are the Methods for Measuring Particle Size of Lipid Nanoparticles?

The particle size of lipid nanoparticles can be measured using different techniques such as dynamic light scattering (DLS), laser diffraction (LD), nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), and various types of applicable microscopy. Among these techniques, DLS is the most commonly used one because it is simple, fast, and non-destructive. DLS measures the Brownian motion of particles in a liquid medium and calculates their hydrodynamic diameter based on the Stokes-Einstein equation. However, DLS also has some limitations, such as relatively low resolution, sensitivity to dust and aggregates, and interference from multiple light scattering, although many DLS manufacturers have made optimizations to address these limitations.

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The Zeta Potential of Lipid Nanoparticles

What is the Zeta Potential of Lipid Nanoparticles?

Zeta potential is defined as the potential of particles on a sliding plane in a liquid medium. It represents the surface charge between particles and the electrostatic repulsion or attraction. Zeta potential affects the colloidal stability, aggregation behavior, flocculation rate, and interaction with biological membranes of lipid nanoparticles. Generally, a higher Zeta potential (positive or negative) indicates stronger electrostatic repulsion and better stability. However, a higher Zeta potential may also increase the toxicity and immunogenicity of lipid nanoparticles. The Zeta potential of lipid nanoparticles is influenced by various factors during their preparation and storage processes, such as lipids, surfactants, solvents, pH, ionic strength, temperature, etc. Depending on the composition and conditions, the Zeta potential of lipid nanoparticles typically ranges from -50 mV to +50 mV.

What are the Methods for Measuring Zeta Potential of Lipid Nanoparticles?

The Zeta potential of lipid nanoparticles can be measured using various techniques such as electrophoretic light scattering (ELS), micro-electrophoresis, electroacoustic spectroscopy, etc. Among these techniques, ELS is the most commonly used one.

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Quality Assays of Lipid Nanoparticles Based on Size & Zeta Potential

Lipid nanoparticles are considered as nanomedicines and must adhere to specific quality standards and undergo rigorous testing before clinical application. The quality standards and detection methods for lipid nanoparticles encompass various aspects including physicochemical properties, biological properties, safety, and efficacy. Some important parameters that must be evaluated and controlled include particle size, zeta potential, morphology, crystallinity, drug loading, drug release, stability, sterility, biocompatibility, cytotoxicity, blood compatibility, immunogenicity, biodistribution, pharmacokinetics, pharmacodynamics, and therapeutic outcomes.

• The standards for particle size and zeta potential of lipid nanoparticles depend on the type, intended use, and regulatory standards. Although there are no universal standards for the optimal particle size and zeta potential of lipid nanoparticles, certain general guidelines can be followed. For instance, for oral administration, the particle size should be less than 500nm to avoid first-pass effect and enhance absorption in the gastrointestinal tract. For intravenous administration, particle size should be less than 200nm to evade rapid clearance by the reticuloendothelial system (RES) and prolong circulation time. For topical administration, particle size should be less than 100nm to enhance penetration into the skin layers.
• Regarding zeta potential, higher values (positive or negative) indicate stronger electrostatic repulsion and better stability of lipid nanoparticles. However, higher zeta potential may also increase the toxicity and immunogenicity of lipid nanoparticles. Hence, a balance between stability and safety should be considered. For instance, for oral administration, a positive zeta potential can enhance mucosal adhesion and absorption of lipid nanoparticles in the gastrointestinal tract. For intravenous administration, a negative zeta potential can reduce the opsonization and phagocytosis of lipid nanoparticles by the RES.

In addition to these general guidelines, specific standards for particle size and zeta potential of lipid nanoparticles are formulated based on different regulatory standards and testing methods. For example, according to FDA regulations, particle size of lipid nanoparticles is measured using dynamic light scattering (DLS) and reported as average size and polydispersity index (PDI). According to European Medicines Agency (EMA) regulations, particle size of lipid nanoparticles is measured using DLS or laser diffraction (LD) and reported as average diameter, PDI, or volume/surface-weighted distribution. According to International Organization for Standardization (ISO) regulations, particle size of lipid nanoparticles is measured using DLS or LD and reported as average diameter or number/volume/surface-weighted distribution. Zeta potential of lipid nanoparticles should be measured using electrophoretic light scattering (ELS) or micro-electrophoresis and reported as mean or distribution. There is no specific threshold for zeta potential of lipid nanoparticles, but it should be justified based on scientific rationale and experimental data.

Lipid nanoparticles are considered as highly promising carriers for bioactive molecules. During preparation and storage, particle size and Zeta potential are two crucial parameters influencing their performance and characteristics. These parameters are regulated by various factors. Quality standards and detection methods for lipid nanoparticles are essential to ensure their quality, safety, and effectiveness in biomedical applications. However, they also pose some challenges and limitations. For instance, deficiencies in standardization and validation, coordination issues among different regulatory bodies and organizations, as well as ethical and environmental considerations. These issues require further development and improvement.