Nanoparticle Size & Distribution Analysis

Accurate nanoparticle size measurement and comprehensive particle size distribution analysis are fundamental requirements for nanoscale research and therapeutic development. Size-dependent properties govern biological interactions, material performance, and regulatory compliance across pharmaceutical, environmental, and materials science applications.

At Hyperion Analytical, our Envision Nanoparticle Tracking Analysis (NTA) system addresses the critical need for precise, reproducible nanoparticle characterization. The technology enables researchers to obtain quantitative size distributions and concentration measurements under physiologically relevant conditions, supporting both fundamental investigations and quality control requirements.

Why Nanoparticle Size Measurement and Distribution Matter

Understanding how nanoparticle size and distribution shape experimental results helps researchers predict performance, ensure consistency, and interpret outcomes with greater confidence.

• Size as a Determinant of Functional Behavior: Even a 10–20 nm change in particle diameter can alter catalytic activity, optical properties, and biological uptake, making nanoparticle size measurement essential across scientific fields.

• Size-Dependent Pharmacokinetics in Drug Delivery Systems: In targeted systems, particles ranging from 80 to 120 nm exhibit optimal tumor accumulation due to enhanced permeability and retention. Smaller ones (<50 nm) clear quickly through the kidneys, while larger particles (>200 nm) are sequestered in the liver and spleen.

• Distribution Characteristics and System Uniformity: Beyond mean diameter, the particle size distribution of nanoparticles indicates population uniformity. A monodisperse population demonstrates predictable, uniform behavior. Polydisperse formulations introduce heterogeneity that compromises performance consistency. Subpopulations within a distribution may exhibit distinct biological fates, which can complicate dose-response relationships and potentially trigger unintended immune responses.

• Regulatory Expectations for Particle Size Characterization: Agencies such as the Food and Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) require consistent particle size distribution data to ensure product stability, reproducibility, and safety throughout a nanomedicine’s lifecycle.

• Environmental Behavior Governed by Particle Dimensions: In natural systems, nanoparticle aggregation, transport, and bioaccumulation are dependent on particle size. Accurate measurement clarifies how these materials behave across changing chemistries and pH conditions.

• Dimensional Influence on Material and Optical Properties: Materials science applications demonstrate equally stringent size requirements. Quantum dots tune fluorescence through size variation, while plasmonic and catalytic nanoparticles show optical and reactivity shifts that scale with particle diameter. Reliable analysis supports reproducible material design.

• Surface Area as a Driver of Reactivity and Interaction: Smaller nanoparticles exhibit exponentially higher surface-to-volume ratios, dramatically influencing dissolution rates, reaction kinetics, and cellular interactions.

• Protein Corona Formation and Its Impact on Measured Size: When nanoparticles enter biological fluids, proteins adsorb onto their surfaces. Smaller particles bind more proteins, altering effective hydrodynamic size and biological identity. Tracking these changes in particle size distribution reveals critical information about nanoparticle stability and uptake pathways.

Challenges in Accurate Nanoparticle Size and Distribution Measurement

Accurately determining nanoparticle size and distribution presents several technical challenges, each of which influences data reliability and interpretation across both research and manufacturing.

Heterogeneity complicates measurement: Real-world samples rarely contain perfectly uniform particles. Polydisperse populations introduce overlapping scattering signals that make it difficult to extract accurate size data using ensemble techniques.

Ensemble methods can mask subpopulations: Techniques like Dynamic Light Scattering (DLS) provide intensity-weighted averages, overrepresenting larger particles and obscuring small but critical populations that influence behavior or stability.

Sample preparation can distort results:  Drying, staining, or immobilizing nanoparticles for microscopy often alters their natural state, especially for soft or hydrated systems such as liposomes or polymeric carriers.

Environmental factors affect diffusion: Temperature, viscosity, and ionic strength all influence Brownian motion. Even slight variations during measurement can lead to inconsistent size estimation if not properly controlled.

Aggregation and sedimentation interfere: In suspension, nanoparticles may aggregate or settle due to gravity, producing misleading distributions. Maintaining stable dispersion is essential for reproducible data.

Limited dynamic range in traditional tools: Many techniques struggle to measure both small nanoparticles and larger aggregates in a single run, leaving gaps in the true particle size distribution of nanoparticles.

Refractive index dependence skews accuracy: Optical methods that rely on light intensity often misrepresent particles with low refractive contrast, such as lipid or polymer nanoparticles, leading to apparent undercounting.

Statistical confidence requires sufficient sampling: Ensemble averages hide variability, while single-particle methods can suffer from a limited field-of-view. The ideal solution provides statistically meaningful data from individual particle tracking.

Benefits of Envision NTA Instruments for Nanoparticle Size and Distribution Analysis

Our Envision NTA instruments address nanoparticle size measurement and distribution challenges efficiently.

• Advanced Optical System: The optical system delivers crisp, clear particle visualization across the entire size range. From 10 nm virus-like particles to 1000 nm liposomes, you get reliable tracking. The temperature control system maintains stability within ±0.1°C, critical for reproducible Brownian motion measurements.

• Fluorescence mode: This mode enables selective tracking of labeled particles within complex mixtures.

• Individual Particle Resolution: In addition to just providing population averages, Envision NTA measures each particle individually, providing true size distributions including subtle subpopulations that other methods might miss.

• Software: Sophisticated software algorithms track thousands of individual particles simultaneously, calculating size from their movement patterns based on the Stokes-Einstein equation. Analysis parameters are transparent and adjustable. With the help of this software, you can see exactly how the algorithm tracks particles and makes size calculations. This is an automated process that requires no manual intervention. The automated analysis routines handle particle tracking, size distribution calculations, and concentration measurements. This level of transparency facilitates both method validation and compliance with regulatory requirements.

• Wide Dynamic Range: These instruments are capable of analyzing particles from 10 nm to 2000 nm in the same sample, accommodating both small therapeutic nanoparticles and larger aggregates or contaminants.

• Low Sample Volume: Our NTA instruments require a few microliters of sample, preserving precious materials and enabling analysis of limited-quantity research samples or clinical specimens.

• Real Concentration Measurement: They simultaneously determine both size distribution and absolute particle concentration, providing complete sample characterization in a single measurement.

• Minimal Sample Preparation: These instruments analyze particles in their native liquid environment without extensive dilution or modification, reducing artifacts and maintaining physiological relevance.

• High-Resolution Distribution: They help resolve complex multimodal distributions and detect minor populations, critical for identifying contaminants, aggregates, or formulation heterogeneity.

• Validation & Reproducibility: Envision NTA delivers highly reproducible results with a coefficient of variation typically under 5% for well-prepared samples. The visual confirmation of particle tracking provides confidence in results, allowing operators to identify and correct potential measurement issues such as air bubbles or contamination.

Key Applications Across Industries

Here are some important application areas of Envision NTA instruments.

• Pharmaceutical Formulation Development: Drug delivery nanoparticles must meet strict specifications. NTA provides both metrics simultaneously and is applicable in various segments in this sector. For instance, liposomal formulations contain multiple lamellae, making them inherently polydisperse NTA resolves individual liposome populations, revealing how formulation variables affect size distribution. You can optimize lipid ratios, production methods, and storage conditions based on real distribution data. For mRNA lipid nanoparticles in vaccines, maintaining the right particle size is critical. Sizes below the optimal range result in rapid clearance, while larger particles exhibited reduced targeting efficiency. NTA ensures quality control by verifying particle size uniformity across batches.

• Nanomedicine: NTA data supports regulatory submissions by providing detailed size distribution profiles and concentration measurements. Stability studies benefit enormously from NTA’s ability to detect early-stage aggregation. They detect even the slightest shift toward larger sizes or a decrease in particle count, which may be warning signs. This enables timely intervention before stability failure occurs. For products with limited shelf life, this predictive capability is invaluable. For generic nanomedicines, matching the average size is not enough. Here, NTA provides the detailed distribution data necessary for these comparisons.

• Virus and Gene Therapy Vectors: Adeno-associated viruses (AAVs), lentiviruses, and other viral vectors represent the future of gene therapy. Characterizing these vectors is challenging. They’re small (typically 20-100 nm), expensive to produce, and their quality directly impacts therapeutic efficacy and safety. NTA quantifies both full and empty capsids based on subtle size differences. This distinction is crucial because empty capsids compete for cell binding without delivering therapeutic genes.

• Exosome and Extracellular Vesicle Research: Exosomes and other extracellular vesicles (EVs) are central to studies of cell communication, disease mechanisms, and emerging therapeutic delivery. Their heterogeneity makes them difficult to characterize using ensemble techniques. Nanoparticle Tracking Analysis (NTA) differentiates small exosomes (50–100 nm) from larger microvesicles (100–1000 nm) and protein aggregates, providing true particle-by-particle size distributions that reflect the biological sample rather than relying on model assumptions.

• Nanomaterial Environmental and Toxicological Studies: NTA enables direct measurement of nanoparticles in complex matrices such as seawater, soil extracts, or biological fluids. It tracks particle aggregation, dissolution, and corona formation as proteins or salts interact with surfaces. These measurements reveal how nanoparticles behave under real-world environmental and physiological conditions, beyond the simplicity of clean laboratory buffers.

• Food Science and Beverage Applications: Food and beverage formulations are increasingly utilizing nanoemulsions and lipid carriers to deliver nutrients and flavors. NTA quantifies particle size and distribution in systems such as milk and coffee, where submicron fat globules and dispersed oils govern texture, flavor, and product consistency.

• Colloid and Surface Chemistry Research: Fundamental studies of colloidal systems benefit from NTA’s ability to track individual particles. Aggregation kinetics, surface charge effects, and stabilization mechanisms all manifest in size distribution changes. By monitoring these changes in real-time, researchers can test theories and develop improved formulations. Gold nanoparticles, quantum dots, and other inorganic nanoparticles often show complex aggregation behavior. NTA reveals how synthesis conditions, surface coatings, and storage conditions affect colloidal stability. This information feeds back into improved synthesis protocols and better-designed nanomaterials.

Achieve Greater Precision in Nanoparticle Characterization

Our continual research and development have helped us develop a new level of capabilities of Envision NTA which aims to further boost nanoparticle analysis. Contact Hyperion Analytical today to learn how Envision NTA can solve your particle size and distribution analysis challenges and support your success in nanotechnology applications.

Frequently Asked Questions (FAQs)

What’s the smallest particle size NTA can reliably measure?

The detection limit depends on particle composition and refractive index. For high refractive index materials like gold or polystyrene, the detection can go as low as 10 -20 nm. For lower refractive index particles like proteins or lipids, 30-50 nm is more typical. The Envision system’s optimized optics push these limits further than most competing instruments.

How does sample viscosity affect NTA measurements?

Higher viscosity slows Brownian motion, which affects size calculations. The Stokes-Einstein equation includes viscosity as a parameter, so you need to know or measure your sample viscosity. For aqueous solutions, this isn’t usually a problem. For organic solvents or high-concentration buffers, viscosity corrections become important.

Can NTA measure nanoparticle concentration in cells or tissue samples?

Direct measurement in intact cells isn’t possible with NTA, which requires particles suspended in liquid. However, NTA excels at measuring nanoparticle uptake after cell lysis or in conditioned media. For internalization studies, cells can be lysed and the lysate analyzed to quantify particle concentration. For tissue biodistribution, homogenized tissue samples may be analyzed, though extensive preparation is needed to separate particles from cellular debris.

Can NTA distinguish between different types of nanoparticles in a mixed sample without fluorescent labels?

Standard scatter-mode NTA cannot chemically distinguish particle types, such as a 100 nm gold nanoparticle and a 100 nm liposome both appear identical if they diffuse at the same rate. However, particles with very different refractive indices scatter light with different intensities, and advanced analysis of scattering intensity distributions can sometimes resolve mixed populations. For definitive identification, fluorescence mode NTA with selective labeling is the preferred approach.