Biophysical Profiling of Protein Corona on Red Blood Cell-Derived Extracellular Vesicles (REVs): Linear Dichroism and...

Extracellular vesicles (EVs) have attracted significant scientific attention due to their critical functions in intercellular communication and their possible uses in diverse fields such as immunology, therapeutics, reproductive biology, biotechnology, and medicine. EVs are engulfed in a layer of proteins, also known as protein corona, which is speculated to play roles in several areas, from intercellular communication through immune recognition to cargo delivery. The composition of the protein corona strongly depends on the origin and the biological environment of EVs. Understanding the protein corona opens doors to finding various applications for vesicles by manipulating them. Typical ways of protein corona removal involve applying high salt concentration or the use of surface-active biomolecules, such as peptides, but this can result in EV membrane damage or complete vesicle disruption. Here, we describe a protocol for characterizing the change in protein corona content on red blood cell-derived EVs (REVs) by using linear dichroism spectroscopy (LD) with microfluidic resistive pulse sensing (MRPS). LD can quantify the change in the amount of the surface attached hemoglobins but cannot identify whether these changes are due to vesicle disruption or protein corona removal from intact vesicles. This necessitates the use of MRPS, which counts the number of vesicles before and after adding a surface manipulating compound. Thus it can identify whether the change in LD signal is due to vesicle disruption or due to loss of protein corona. The combined methods can be employed to understand which process takes place in which ratio, allowing the optimization of vesicle engineering toward specific needs.