(734f) Single Lipoplex Fluorescent Nanoparticle Tracking Analysis and Applications for the Study of Exosomes

The study of exosomes, 40-100nm lipid bodies released by virtually all cells in the body, has seen tremendous growth in recent years. Long considered artifacts of cellular processing, new results have revealed that these bodies not only carry important cargo, but have the ability to interact specifically with surrounding cells and elicit functional changes in the target. Recent research has identified exosomes as key vehicles of intercellular communication, and characterization of the pathways and machinery they utilize to transfer information between cells is a rapidly expanding research field.  Beyond cellular communication a number of studies have generated interest in exosomes as biomarkers for a variety of human diseases including cancers, Alzheimer’s disease, HIV, and kidney disorders.  The success of liposome encapsulated therapeutics has also led to proposals for producing therapeutic exosomes, wherein a desired cargo will be completely produced, enclosed in an exosome, and separated from a culture supernatant. With the explosion of interest in this field, the development of reliable techniques for isolating and characterizing exosomes is necessary and relevant.  To date more than four thousand proteins and two thousand mRNAs have been identified in exosomes, however many studies have relied on the examination of hetergenous bulk exosome populations to produce results, leading to obscuration of markers expressed at low levels or in specific smaller subpopulations.  Moving forward, the ability to identify specific proteins and RNAs at the single exosome level will be required, along with reliable methods of assessing separation schemes for isolating specific subpopulations from bulk exosome preparations.  This presentation examines the application of nanoparticle tracking analysis (NTA) to identify and track single fluorescently labeled nanoparticles for both characterization and isolation purposes.

Nanoparticle Tracking Analysis (NTA) is a widely used technique for determining the size and number concentration of various submicron particles, including catalyst particles, viruses, protein aggregates, in addition to exosomes. NTA allows for the visualization and sizing of single nanoparticles in suspension by observing their Brownian motion from light scattering methods. However, impure preparations and lack of specific signaling can make this analysis prone to errors from contaminating particles in the exosome preparation.   NTA can also track fluorescently labeled nanoparticles in the same manner, and this method in conjunction with an appropriate labeling method can provide a confirmation of particle identity. Herein we demonstrate the effect of fluoroprobe loading in self assembling lipoplex (anionic nucleic acid – cationic lipid complexes) particles on fluorescent NTA size and concentration measurements when compared to light scattering measurements of the same particles. Understanding the difference between these two methods and the associated phenomena is critical to proper application of NTA to the study of fluorescently labeled exosomes.   In addition the lipoplex particles employed in this work have the ability to bind exosomes, and we demonstrate a method using NTA for determining the exosome binding capacity of these particles, which will find application in future techniques for separating and characterizing specific subpopulations of exosomes. The results of this presentation provide a method for determining the lower limit of fluorescence sensitivity in an NTA system, a value which must be known for effective fluorescent labeling of exosomes, an understanding of how measured results will differ between fluorescent and scattering observations, and a method of quantifying the exosome binding ability of particles for separations.