Nanoscopic Profiling of Small Extracellular Vesicles via High-Speed Atomic Force Microscopy (HS-AFM) Videography

Area of Science:

• Biophysics
• Cell Biology
• Nanotechnology

Background:

• Small extracellular vesicles (sEVs) are crucial for intercellular communication and serve as potential biomarkers.
• Current purification methods yield heterogeneous sEV populations, limiting detailed analysis.
• Understanding sEV heterogeneity is vital for accurate biomarker interpretation and therapeutic applications.

Purpose of the Study:

• To investigate the intracellular origins and heterogeneity of small extracellular vesicles (sEVs) at single-vesicle resolution.
• To explore the feasibility of using high-speed atomic force microscopy (HS-AFM) with exosome markers for sEV characterization.
• To differentiate between distinct sEV subpopulations based on surface marker expression and physical properties.

Main Methods:

• Utilized high-speed atomic force microscopy (HS-AFM) to image HEK293T-derived sEVs under physiological conditions.
• Employed exosome marker antibodies (IgGCD63 and IgGCD81) to probe surface protein localization.
• Analyzed nanotopology, structural dynamics, and marker co-localization on individual sEVs.

Main Results:

• HS-AFM revealed distinct nanotopological features and height fluctuations between larger (diameter > 100 nm) and smaller (diameter ≤ 100 nm) sEVs.
• Exosome marker antibodies (IgGCD63, IgGCD81) showed predominant co-localization with smaller sEVs (sEVd ≤ 100 nm).
• Demonstrated the existence of CD63-CD81-enriched and CD63-CD81-depleted sEV subpopulations within a heterogeneous mixture.

Conclusions:

• Nanoscopic profiling of surface exosome markers on sEVs using HS-AFM is a feasible method for characterizing distinct sEV subpopulations.
• This approach enables the differentiation of heterogeneous sEV mixtures, paving the way for more precise biomarker discovery.
• The findings highlight the potential of HS-AFM in advancing the understanding of sEV heterogeneity and function.