Detection by super-resolution microscopy of viral proteins inside bloodborne extracellular vesicles.

AIM

Extracellular vesicles (EVs) are small particles released by all cells, including virally infected cells, into the extracellular space. They play a role in various cellular processes, including intercellular communication, signaling, and immunity, and carry several biomolecules like proteins, lipids, and nucleic acids that can modulate cellular functions mostly by releasing their cargo inside the target cells via the endocytic pathway. One of the most exciting aspects of EV physiology is its potential in liquid biopsy as a diagnostic and prognostic marker. However, due to their extremely small size and lack of a molecular approach to examine intravesicular content or cargo, we cannot fully utilize their potential in healthcare.

METHODS

Here, we present a novel approach that allows examining bloodborne EVs at a single-particle level with the ability to examine their cargo without disrupting structural integrity. Our technique utilizes super-resolution microscopy and a unique permeabilization process that maintains structural integrity while facilitating the examination of EV cargo. We used a mild-detergent-based permeabilization buffer that protects the integrity of EVs, minimizes background, and improves detection.

RESULTS

Utilizing this approach, we were able to recognize viral proteins of SARS-CoV-2 virus in COVID-19 patients, including spike and nucleocapsid. Surprisingly, we found an almost equal amount of spike protein inside and on the surface of bloodborne EVs. This would have proven difficult to determine using other conventional methods.

CONCLUSION

To summarize, we have developed an easy-to-perform, sensitive, and highly efficient method that offers a mechanism to examine bloodborne EV cargo without disrupting their structural integrity.