Quantitative and qualitative comparison of mRNA loading techniques into extracellular vesicles

Area of Science:

• Biotechnology
• Nanomedicine
• Molecular Biology

Background:

• Extracellular vesicles (EVs) show promise as natural therapeutic delivery systems (DDS).
• Efficiently loading sensitive biomolecules like messenger RNA (mRNA) into EVs remains a significant challenge.
• Inconsistent literature data hinders the development of reliable EV-based mRNA delivery platforms.

Purpose of the Study:

• To systematically compare five exogenous methods for loading mRNA into EVs.
• To quantitatively assess loading efficiency, biophysical, biochemical, and functional properties of mRNA-loaded EVs.
• To identify optimal methods for advancing EV-based mRNA therapeutics.

Main Methods:

• Comparison of five mRNA loading techniques: incubation, sonication, freeze-thaw, electroporation, and extrusion.
• Utilized direct stochastic optical reconstruction microscopy (dSTORM) for visualizing and quantifying single mRNA molecules within single EVs.
• Evaluated loading efficiency, EV integrity, and mRNA association.

Main Results:

• All five methods showed high EV association with mRNA (up to 70%), with minimal disruption to EV properties.
• Freeze-thaw demonstrated the highest loading efficiency and greatest amount of associated mRNA per EV.
• Despite high association, overall mRNA incorporation efficiency was limited, with significant mRNA loss during loading.

Conclusions:

• Freeze-thaw and electroporation show promise for increasing mRNA association with EVs.
• Exogenous mRNA loading into EVs faces challenges for large-scale implementation due to mRNA loss.
• This study provides a foundation for optimizing mRNA loading in EV-based DDS for therapeutic applications.