Nanoparticle Tracking Analysis for Characterizing Exosomes

Exosomes are nanoscale vesicles released by cells that facilitate cell-to-cell communication by transporting molecules like proteins and RNA. Their presence in bodily fluids is linked to various health and disease states, making them candidates for new diagnostic and therapeutic strategies. A challenge lies in accurately measuring these particles. Nanoparticle Tracking Analysis (NTA) is a method for determining the physical properties of exosomes, providing data on their size and concentration to advance research from the laboratory to clinical applications.

The Principles of Nanoparticle Tracking Analysis

Nanoparticle Tracking Analysis operates by visualizing the movement of individual nanoparticles in a liquid. The system uses a sample chamber where the liquid is illuminated by a focused laser beam. When particles pass through this beam, they scatter light, which is captured by a microscope connected to a high-sensitivity digital camera. The camera records a video file of the real-time movement of each visible particle.

This movement follows the principle of Brownian motion, the erratic movement of particles in a fluid from collisions with surrounding molecules. The NTA software tracks the trajectories of individual particles from the video footage. By analyzing these paths frame by frame, the software calculates how far each particle moves over a given period.

From this movement, a diffusion coefficient is determined for each particle. Smaller particles move more rapidly than larger ones, resulting in a higher diffusion coefficient. This coefficient is used in the Stokes-Einstein equation, which relates the diffusion coefficient to particle size. The software then calculates the hydrodynamic diameter of each particle, generating a size distribution profile for the sample.

Characterizing Exosomes with NTA

When applied to exosomes, NTA provides two primary measurements: particle concentration and size distribution. Particle concentration is reported as the number of exosome particles per milliliter of the sample, offering a quantitative measure of their abundance. This is determined by counting the tracked particles within a known volume of the sample chamber. This metric is used for standardizing experiments and comparing exosome secretion levels between different cell types or conditions.

The second output is the size distribution profile, presented as a graph showing the range of particle diameters and the frequency of each size. Exosomes fall within a size range of 30 to 150 nanometers, and the NTA plot can verify the presence of a population within this range. The shape of this distribution can also offer insights into the purity of the sample, as particles outside the typical exosome range might indicate contamination with other extracellular vesicles or debris.

Fluorescence NTA (F-NTA) is an advanced application that enhances specificity. In this mode, exosomes are labeled with fluorescent molecules, such as antibodies tagged with dyes that bind to specific protein markers on the exosome surface. The instrument is then configured to track and analyze only the particles that emit a fluorescent signal. This allows it to specifically measure the concentration and size of a targeted subpopulation, distinguishing genuine exosomes from other similarly sized particles.

Preparing Exosome Samples for Analysis

The quality of data from Nanoparticle Tracking Analysis depends on proper sample preparation. A primary step is sample dilution, as the concentration of exosomes in samples from cell culture or biological fluids is often too high for the instrument to analyze accurately. If particles are too concentrated, their light-scattering signals can overlap, preventing the software from distinguishing individual particles.

The sample must be diluted to an optimal concentration range, typically between 10^7 and 10^9 particles per milliliter. The choice of diluent is also a consideration; filtered phosphate-buffered saline (PBS) is commonly used as it is compatible with biological samples. The diluent must be meticulously filtered to remove any pre-existing contaminants that could be mistakenly counted.

Another preparatory action involves removing unwanted components from the sample. Larger particles, such as cell debris or large protein aggregates, can scatter much more light than exosomes, obscuring the signals from the smaller particles of interest. To prevent this, samples are often subjected to low-speed centrifugation or filtration to remove larger contaminants and ensure that only particles within the desired size range enter the instrument.

Applications in Diagnostics and Therapeutics

The measurements from NTA have direct applications in medicine, particularly in diagnostics and therapeutic development. In diagnostics, NTA is used to analyze exosomes in patient biofluids, such as blood or saliva, in a process known as a “liquid biopsy.” The concentration and size of exosomes can change in the presence of diseases like cancer. By quantifying these changes with NTA, it may be possible to develop non-invasive tests for early disease detection.

In the development of new medicines, exosomes are engineered to act as drug delivery vehicles, carrying therapeutic cargo to target cells. NTA serves as a quality control tool during the manufacturing process. It is used to ensure that each batch of exosome-based therapeutics meets specifications for particle concentration and size distribution. This consistency helps guarantee that each dose delivers the intended therapeutic effect and adheres to safety standards.