Contaminating transfection complexes can masquerade as small extracellular vesicles and impair their delivery of RNA

Abstract

One of the functions of small extracellular vesicles (sEVs) which has received the most attention is their capacity to deliver RNA into the cytoplasm of target cells. These studies have often been performed by transfecting RNAs into sEV‐producing cells, to later purify and study sEV delivery of RNA. Transfection complexes and other delivery vehicles accumulate in late endosomes where sEV are formed and over 50% of transfection complexes or delivery vehicles administered to cells are released again to the extracellular space by exocytosis. This raises the possibility that transfection complexes could alter sEVs and contaminate sEV preparations. We found that widely used transfection reagents including RNAiMax and INTERFERin accumulated in late endosomes. These transfection complexes had a size similar to sEV and were purified by ultracentrifugation like sEV. Focusing on the lipid‐based transfection reagent RNAiMax, we found that preparations of sEV from transfected cells contained lipids from transfection complexes and transfected siRNA was predominantly in particles with the density of transfection complexes, rather than sEV. This suggests that transfection complexes, such as lipid‐based RNAiMax, may frequently contaminate sEV preparations and could account for some reports of sEV‐mediated delivery of nucleic acids. Transfection of cells also impaired the capacity of sEVs to deliver stably‐expressed siRNAs, suggesting that transfection of cells may alter sEVs and prevent the study of their endogenous capacity to deliver RNA to target cells.

Evaluation of EV Storage Buffer for Efficient Preservation of Engineered Extracellular Vesicles

“…Our data that extracted values of larger particles (>200 nm) presented evidence of particle fusion or aggregation within the PBS buffer with statistical means in both fresh and preserved samples (Figure 2C). Yet, these measures lack validation of individual EVs that cannot exclude the possibility of counting non-EV particles, such as transfection complex 32 , protein aggregates, or serum byproducts. Nevertheless, these measures from the current study could serve as one indicator of size variation among easily adaptable storage conditions.…”

Advances in Therapeutic Applications of Extracellular Vesicles

“… 173 However, transfection changes the genetic material contained in EVs, which may impair the ability of EVs to deliver stably expressed siRNA and even raise potential biosafety problems. 174 , 175 Electroporation, on the other hand, uses electric fields to create temporary hydrophilic pores in the phospholipid membrane of exosomes and load hydrophilic biomaterials. This technology is widely applicable and has been used to successfully deliver Adriamycin (for increased targeting) and doxorubicin (an antitumor agent) into MSC-EXO to TUBO breast cancer cell lines, reducing tumor growth rates, while free Adriamycin and non-targeted doxorubicin exosomes had no effect.…”

Novel Endogenous Engineering Platform for Robust Loading and Delivery of Functional mRNA by Extracellular Vesicles

“…Depending on the nature of the RBD, such as whether it recognizes a structural element or specific nucleotide sequences, corresponding interacting sites are inserted in the 3´UTRs of the mRNA (Targeted and Modular EV Loading (TAMEL) approach) 26 . Substantial drawbacks of current endogenous EV engineering strategies for mRNA loading include mRNA instability due to long artificial 3'UTRs, insufficient mRNA loading despite active EV sorting, as well as unsolicited carry-over of plasmid DNA that potentially interferes with the assessment of mRNA functionality [29][30][31] . To address these shortcomings, we developed a novel TAMEL-based platform for efficient mRNA delivery using engineered EVs.…”

“…Substantial drawbacks of current endogenous EV engineering strategies for mRNA loading include mRNA instability due to long artificial 3’UTRs, insufficient mRNA loading despite active EV sorting, as well as unsolicited carry-over of plasmid DNA that potentially interferes with the assessment of mRNA functionality 29–31 . To address these shortcomings, we developed a novel TAMEL-based platform for efficient mRNA delivery using engineered EVs.…”