Digging Deeper into Nanoscale: Size-Based Extracellular Vesicle Characterization with Taylor Dispersion Analysis

Introduction

Extracellular vesicles (EVs) are of great interest in clinical research among many other applications due to their diverse roles in biological processes and their potential implications for various diseases. They can contain a variety of molecules that reflect the physiological state of the cell they originated from. This makes them attractive as potential biomarkers for various diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders. Furthermore, EVs can be engineered to deliver therapeutic molecules, such as drugs or nucleic acids, to target cells. This approach has the potential to improve drug delivery efficiency, reduce side effects, and enhance treatment outcomes. The size and the variability of extracellular vesicles require characterization equipment able to detect particles from 0.5 to 200 nm. Taylor dispersion analysis uses the understanding of the motion of objects, including biosamples, in a laminar flow to characterize their size by calculating their radial diffusion coefficient. Based on this principle, we were able to characterize the FluoEV samples using the Taylorsizer.

EV Preparation

This study included our CD63-EGFP FluoEVs (Product code: HBM-HEK-EGFP63).

FluoEVs are products of genetic EV engineering to their parents, HEK293 cells. As a result of this genetic engineering, they carry fluorescent EGFP, BFP, or mCherry proteins in fusion with well-known EV markers: CD9, CD63, and CD81. This way, they emit fluorescence stably without photobleaching or non-specific background signal problem.

FluoEVs are provided in lyophilized form and reconstituted simply by adding MilliQ water.

Characterization

Two formulations of FluoEVs were characterized by SD-TDA with an excitation at 488nm. Experiments were performed on a TaylorSizer instrument by Nanoscale Metrix with a fluorescent detector. The experiments were performed on a bare fused silica capillary with inner diameter 50 μm, total length 60 cm, and length to detection window 49.5 cm. The temperature of the capillary area was set at 25°C. SD-TDA experiments were performed using an in-house developed protocol allowing for the injection of only 20 nL.

The Taylorgrams indicate the presence of aggregates with spikes that show up before the sample peak. In the analysis of the size distribution of the particles, the sample is mostly made up of extracellular vesicles with an average size of 80nm. However, two other nanoparticles subgroups with peaks centered at 25nm and 5nm are also found. Even though further marker analyses are necessary to fully characterize these subgroups, these peaks can be attributed to a little presence of small nanoparticles as exomeres, and supermeres or some membrane fragments.

Conclusion

This study demonstrates the efficacy of Size Distribution Taylor Dispersion Analysis (SD-TDA) for the characterization of extracellular vesicles (EVs). By utilizing the TaylorSizer, we were able to accurately determine the size distribution of FluoEVs samples, revealing a major population with typical diameter of small EVs (50 – 100 nm) and two minor subpopulations with diameter <20nm, which can be attributed to the presence of some small nanoparticles such as exomeres, supermeres, and biological residue. The presence of aggregates and the variability in particle size highlight the importance of a precise characterization technique for understanding the heterogeneity of EV populations and their potential implications in various biological processes and disease states.

SD-TDA offers a valuable tool for researchers investigating EV-based applications. The ability to accurately characterize EV size under 50 nanometers is essential for optimizing the isolation, purification, and functionalization of these nanoparticles, paving the way for advancements in personalized medicine and targeted therapies.