Lipid Nanoparticle Analysis

Comparing Analytical Techniques for Lipid Nanoparticle Analysis

Multiple analytical techniques exist for LNP characterization, each with distinct capabilities and limitations. Here are a few popular ones among them.

• Dynamic Light Scattering (DLS): DLS measures intensity fluctuations from many particles to estimate size distributions quickly and with minimal sample volume. However, its intensity-weighted output favors larger particles (signal scales with the sixth power of diameter), masking minor populations or aggregates. The resulting z-average diameter yields a single mean value but provides little insight into heterogeneity, making DLS less reliable for complex or polydisperse lipid nanoparticle formulations.

• Transmission Electron Microscopy (TEM): Electron microscopy provides direct visualization of individual nanoparticles with exceptional resolution. Structural details and morphology become apparent. However, sample preparation, such as drying, staining, or vitrification, can introduce artifacts. The high vacuum environment differs substantially from native aqueous conditions. Statistical sampling is limited because only a few hundred particles can be practically analyzed, compared to thousands or millions by other techniques. TEM excels at confirming particle morphology but may not accurately represent the true size distribution in solution.

• Atomic Force Microscopy (AFM): AFM generates nanoscale topographical maps by scanning a sharp probe across individual particles. This lipid nanoparticle characterization technique provides precise height and surface morphology measurements without relying on optical contrast. However, samples must be immobilized on solid substrates, often in dry conditions, which can distort soft lipid nanoparticles. Imaging throughput is low, covering only small areas. AFM excels at visualizing surface structure but offers limited statistical insight into population-wide size distributions.

• Differential Centrifugal Sedimentation (DCS): DCS separates particles based on their sedimentation rate through a fluid with a known density gradient, providing high-resolution size distributions and excellent sensitivity to slight differences in diameter. It is particularly effective for monodisperse samples. However, accurate results depend on precise density and viscosity inputs, and irregularly shaped or soft lipid nanoparticles may behave unpredictably. Sample preparation and calibration can be time-consuming, limiting routine throughput.

• Nanoparticle Tracking Analysis: Nanoparticle tracking analysis addresses many limitations of alternative lipid nanoparticle characterization techniques while providing unique advantages for lipid nanoparticle analysis. The method tracks individual particles in solution as they undergo Brownian motion, capturing both size and concentration information simultaneously.

NTA operates in near-native conditions with minimal sample preparation. Particles remain in liquid suspension at physiological pH and ionic strength. This preserves particle structure and aggregation state. The particle-by-particle approach means each particle contributes equally to the size distribution regardless of size. Polydisperse samples are characterized accurately without mathematical biasing toward larger particles.

The technique detects and quantifies subpopulations that ensemble methods might miss. Sample requirements are modest (typically 200 microliters at 10^9 to 10^12 particles/mL). With analysis completed in only 3–5 minutes, researchers can screen multiple formulations efficiently during process development and quality control.

Size resolution allows discrimination of populations separated by 20-30 nanometers. The measurable range (approximately 30-1000 nm) encompasses typical LNP formulations plus larger aggregates. Concentration measurements provide directly useful metrics for dose calculations without requiring standard curves or calibration factors.

NTA provides an optimal balance of information content, throughput, and practical usability for researchers developing lipid nanoparticles for drug therapy or other pharmaceutical applications. The technique complements orthogonal methods, such as TEM for morphology or cryo-EM for structure, while serving as a primary tool for routine characterization, quality control, and process monitoring.

How Envision NTA Analyzes Lipid Nanoparticles

The Envision system from Hyperion Analytical implements lipid nanoparticle tracking analysis with features specifically designed for pharmaceutical research and quality control applications.

• Envision NTA directly visualizes lipid nanoparticles in suspension, tracking individual particles as they undergo Brownian motion to determine their size and concentration simultaneously.
• Each particle’s motion is analyzed using the Stokes–Einstein relationship, giving an absolute hydrodynamic diameter without needing external calibration or assumptions about refractive index or density.
• Measurements are performed in liquid under near-physiological conditions, preserving the true structure and aggregation state of lipid nanoparticles for accurate, representative analysis.
• Envision NTA’s lower practical range and upper practical range for lipid nanoparticles is about 30nm to 1000 nm. However, 1000 nm is not ideal for soft LNPs. This wide range allows detection of both small vesicles and larger aggregates that often form in complex LNP formulations.
• Envision’s fluorescence mode isolates fluorescently labeled lipid nanoparticles from background scatter, enabling tracking of specific populations, such as targeted or encapsulated particles, in mixed samples.
• The system provides absolute particle concentration in particles per milliliter, offering directly useful data for dose determination, formulation consistency, and process development.
• A precisely engineered optical design ensures stable illumination and minimal background noise, resulting in high signal-to-noise images and consistent tracking even for weakly scattering LNPs.
• Automated alignment and controlled flow maintain reproducibility across runs, reducing user variability and ensuring reliable size and concentration measurements over time.
• The operation of Envision NTA for lipid nanoparticle analysis is quite straightforward. It follows a simple pipette loading, a sealed flow cell, and an integrated syringe pump that prevents bubbles or leaks. Cleaning requires only a brief rinse; no disassembly is needed.
• With analysis times of only minutes, Envision NTA enables quick, quantitative evaluation of lipid nanoparticle quality and stability for research and manufacturing.

Key Benefits of Using NTA for Lipid Nanoparticle Analysis

The following key benefits highlight why Nanoparticle Tracking Analysis (NTA) has become an essential tool for accurate, efficient, and insightful lipid nanoparticle characterization across research and manufacturing settings.

• High-Resolution Size Distributions: NTA tracks individual particles, generating number-weighted size distributions free from bias toward larger particles. It resolves subpopulations separated by 20–30 nm, detecting subtle formulation changes or early aggregation that ensemble methods often miss.

• Simultaneous Size and Concentration Measurement: Size and concentration data are obtained from one analysis, reducing sample use and time. Results in particles per milliliter directly inform dose and formulation stoichiometry, minimizing variability from separate assays.

• Near-Native Measurement Conditions: During lipid nanoparticle analysis using NTA, the particles are analyzed in liquid suspension without drying or labeling, maintaining their true structure and aggregation state. Measurements under physiological or stress-test conditions yield data representative of real-world performance and storage stability.

• Practical Sample Requirements: For NTA, only ~200 µL of sample is needed, making this technique ideal for limited material in early research. The label-free method relies on natural light scattering, simplifying workflows and avoiding changes in particle behavior caused by fluorescent tags.

• Rapid Analysis for Process Development: Typical NTA measurements take 3–5 minutes, enabling daily screening of multiple formulations. Researchers can efficiently optimize lipid ratios, PEG content, or encapsulation methods across 15–25 samples per day.

• Sensitive Aggregate Detection: NTA captures particles in the 30–1000 nm range, enabling early detection of 1–5% aggregate populations. This helps prevent stability problems before they affect product quality.

• Quality Control Applications: NTA provides quantitative metrics for batch consistency, including mean size, distribution width, and aggregate content, supporting release criteria and validation under ICH guidelines.

• Regulatory Support: Recognized by regulators for nanomedicine characterization, NTA’s physics-based method (Brownian tracking, Stokes–Einstein) and validated parameters align with submission requirements.

• Troubleshooting Capabilities: Detailed distributions of NTAs help pinpoint causes of failed formulations or process deviations, guiding rapid corrective actions and conserving resources.

Key Applications of Lipid Nanoparticles

Lipid nanoparticle technology enables the delivery of therapeutic molecules that would otherwise be unstable or unable to reach their cellular targets. Each application area presents specific analytical requirements that comprehensive lipid nanoparticle analysis addresses.

• mRNA Vaccines and Therapeutics: mRNA lipid nanoparticles (mRNA-LNPs) protect RNA from enzymatic degradation and facilitate intracellular delivery. Particle sizes of 60–100 nm determine transfection outcomes and biodistribution profiles. NTA enables precise characterization of mRNA-LNP formulations, supporting optimization, aggregation monitoring, and stability assessment during storage.

• siRNA and Gene Silencing Therapies: siRNA-loaded LNPs require stability and targeting precision. Surface ligands like GalNAc enhance tissue selectivity but can affect size and aggregation. NTA verifies formulation integrity, ensuring surface modifications maintain critical parameters while supporting consistent manufacturing and optimization of siRNA therapeutics.

• Gene Editing Technologies: CRISPR-Cas9 and related tools rely on lipid nanoparticles for gene delivery of mRNA, proteins, or ribonucleoprotein complexes. NTA evaluates how these large cargos influence particle formation, size, and stability. It helps optimize encapsulation efficiency and track degradation, which is crucial for less stable ribonucleoprotein-based formulations.

• Cancer Immunotherapy: LNPs deliver tumor antigens, adjuvants, or immune modulators to targeted cells, influencing immune activation and biodistribution. NTA ensures uniform particle size and stability in multi-component formulations, confirming that co-loaded agents do not aggregate or destabilize the lipid matrix.

• Protein and Peptide Delivery: Encapsulating proteins or peptides in LNPs improves stability and controlled release but can alter particle behavior. NTA verifies the uniformity of encapsulation and detects aggregation. Applications include enhanced oral delivery of peptides like insulin and enzyme replacement therapies for intracellular delivery.

• Rare Genetic Disease Treatments: For small patient populations, LNP-based mRNA or gene editing therapies must meet rigorous quality standards. NTA ensures batch consistency, verifies encapsulation performance, and provides data supporting regulatory documentation for therapies targeting single-gene disorders.

• Infectious Disease Vaccines: Beyond COVID-19, mRNA-LNP vaccines are in development for influenza, respiratory syncytial virus (RSV), cytomegalovirus, and other infectious diseases. NTA supports both early development and large-scale manufacturing, providing consistent particle-size and concentration analysis across research, clinical, and commercial production batches.

Get Clear, Quantitative Insight into Your Lipid Nanoparticle Characterization

Connect with Hyperion Analytical to see how Envision NTA simplifies LNP characterization, delivering precise size and concentration data in minutes. Request a consultation or arrange a sample evaluation with our applications team.

Frequently Asked Questions

Can NTA distinguish between empty and cargo-loaded LNPs?

NTA primarily measures hydrodynamic size based on Brownian motion. Empty versus loaded particles might show size differences if cargo substantially affects particle diameter. However, NTA doesn’t directly measure cargo content or loading efficiency. Combining NTA with complementary techniques (such as fluorescence correlation spectroscopy, analytical ultracentrifugation, or UV spectroscopy for nucleic acid quantification) provides more complete characterization, including encapsulation efficiency.

What sample concentrations are optimal for NTA measurements?

Optimal concentrations in the measurement chamber range from 10^7 to 10^9 particles/mL. The Envision system includes automated dilution, so stock LNP formulations at 10^10 to 10^12 particles/mL can be measured directly. Higher concentrations cause tracking errors from overlapping particle images. Lower concentrations provide insufficient particles for statistical significance. The software provides real-time feedback on whether concentration is appropriate for reliable tracking.

Can LNPs be measured in complex biological media?

Complex fluids, such as serum, plasma, and cell culture media present challenges because they contain proteins, lipoproteins, and other components that scatter light. Measurements are possible if LNPs are sufficiently larger than background components or if samples are diluted to reduce background interference. Fluorescently labeled LNPs enable specific detection in complex media by tracking fluorescence rather than scattered light. For most formulation development and quality control work, measurements in simpler buffers provide better data quality and reproducibility.

What size range can NTA reliably detect for lipid nanoparticles?

The lower detection limit depends on the particle’s refractive index and the instrument’s sensitivity. For lipid nanoparticles, reliable detection typically begins around 30-40 nm. Smaller particles scatter insufficient light for consistent tracking. The upper limit is approximately 1000 nm, though substantial particles sediment rather than diffuse solely by Brownian motion. Typical LNP formulations (60-150 nm) fall well within the optimal measurement range where tracking accuracy is highest.

How do buffer conditions affect NTA measurements?

Buffer composition affects particle properties and measurement quality. Viscosity influences diffusion rates and, therefore, calculated sizes; the instrument compensates for known viscosities. Ionic strength affects particle surface charge and aggregation state. High concentrations of particulate contaminants create background scatter that interferes with tracking. Most pharmaceutical buffers, such as PBS, HEPES, citrate, and acetate, work well for NTA. Temperature control is essential because viscosity and diffusion are temperature-dependent; the Envision system maintains precise temperature during measurement.

Can NTA measurements be automated for high-throughput screening?

The Envision system includes features supporting efficient workflows. Automated sample introduction via syringe pump eliminates manual injection variability. Pre-programmed measurement sequences run with minimal operator intervention. Current configurations handle 15-25 samples per day comfortably, which suffices for most quality control and formulation screening applications. For applications requiring higher throughput like hundreds of samples daily, custom automation solutions can be developed. The balance between thoroughness (tracking thousands of particles per sample) and speed determines practical throughput limits.