Quantitative and qualitative comparison of mRNA loading techniques into extracellular vesicles

Extracellular vesicles (EVs) are increasingly recognized as natural therapeutic delivery systems (DDS). A critical challenge in realizing this potential is the loading of nucleic acids, particularly mRNA, a high molecular weight and very sensitive biomolecule, for which the loading efficiency into EVs is often low. Despite the growing interest in EV-based delivery platforms, relatively few studies have reported efficient loading techniques, and inconsistent or misleading information in the literature hinders progress towards the development of reliable loading of mRNA into EV-based systems. Addressing this gap systematically and quantitatively is crucial for the clinical translation of EVs as DDS. In this study, we compared five different exogenous mRNA loading methods (incubation, sonication, freeze-thaw, electroporation and extrusion) and evaluated their loading efficiencies, as well as their biophysical, biochemical, and functional properties. Here, using direct stochastic optical reconstruction microscopy (dSTORM), as an efficient and reliable characterization approach to assess mRNA loading efficiency into EVs we visualized single EVs and single mRNA molecules and to estimate the percentage of loaded EVs as well as the amount of mRNA molecules loaded per EV. Interestingly, all five strategies demonstrated a high yield of EVs associated with mRNA, with up to 70 % of EVs successfully containing or in contact with mRNA, with minimal disruption to the intrinsic properties of EVs. Although some discrepancies between loading efficiencies, functionality and amount of mRNA molecules per EV, freeze-thaw proved to be the most effective in terms of loading efficiency and amount of mRNA associated molecules per EV. Overall, mRNA incorporation using these exogenous methods remained inefficient and presented significant challenges for large-scale implementation due to substantial mRNA loss. Despite this limitation, the high proportion of successfully loaded or mRNA associated EVs, particularly with freeze-thaw and electroporation, provides, for the first time, a promising foundation for the advancing of mRNA loading on EV-based therapeutic applications.