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

Jenna McCann; Carmen Daniela Sosa‐Miranda; Huishan Guo; Ryan Reshke; Alexandre Savard; Adriana Zardini Buzatto; James A. Taylor; Liang Li; Derrick J. Gibbings

doi:10.1002/jev2.12220

journal article Open Access Oct 01, 2022

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

Jenna McCann Carmen Daniela Sosa‐Miranda Huishan Guo Ryan Reshke Alexandre Savard Adriana Zardini Buzatto

James A. Taylor Liang Li

Derrick J. Gibbings

Journal of Extracellular Vesicles Vol. 11 No. 10 · Wiley

View at Publisher Save 10.1002/jev2.12220

Abstract

AbstractOne 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.

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References

75

[1]

10.1056/nejmoa1716153

[2]

10.1101/cshperspect.a016980

[3]

10.1038/mt.2010.85

[4]

10.1038/nbt.1807

[5]

10.1056/nejmoa1913147

[6]

Cellular uptake of nanoparticles: journey inside the cell

Shahed Behzadi, Vahid Serpooshan, Wei Tao et al.

Chemical Society Reviews 10.1039/c6cs00636a

[7]

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane

Amy M. Booth, Yi Fang, Jonathan K. Fallon et al.

The Journal of cell biology 10.1083/jcb.200508014

[8]

10.1093/nar/gkaa670

[9]

10.3390/ph14040356

[10]

10.1371/journal.pone.0021503

[11]

10.1073/pnas.1408301111

[12]

10.1038/mt.2016.126

[13]

10.1038/nbt.3802

[14]

10.1038/s41467-020-15300-1

[15]

10.1002/adhm.202101202

[16]

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Andrew Fire, SiQun Xu, Mary K. Montgomery et al.

Nature 10.1038/35888

[17]

10.1093/nar/gkv762

[18]

Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape

Jerome Gilleron, William Querbes, Anja Zeigerer et al.

Nature Biotechnology 10.1038/nbt.2612

[19]

CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis

Julian D. Gillmore, Ed Gane, Jorg Taubel et al.

New England Journal of Medicine 10.1056/nejmoa2107454

[20]

10.1126/science.8209258

[21]

10.1038/s41578‐021‐00358‐0

[22]

10.1016/s0006-3495(96)79309-8

[23]

Reassessment of Exosome Composition

Dennis K. Jeppesen, Aidan M. Fenix, Jeffrey L. Franklin et al.

Cell 10.1016/j.cell.2019.02.029

[24]

Jonker C. "An adapted protocol to overcome endosomal damage caused by polyethylenimine (PEI) mediated transfections" Matters (2017)

[25]

10.1021/acsnano.9b10033

[26]

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer

Sushrut Kamerkar, Valerie S. LeBleu, Hikaru Sugimoto et al.

Nature 10.1038/nature22341

[27]

10.1038/nmat3765

[28]

10.1038/mt.2011.294

[29]

10.1002/jgm.173

[30]

10.1126/sciadv.abf4398

[31]

10.1016/j.ymthe.2021.02.015

[32]

10.1038/s41467-018-03733-8

[33]

10.1016/j.jconrel.2013.08.014

[34]

10.1021/acsnano.8b01516

[35]

10.1016/s0022-1759(02)00330-7

[36]

10.1038/s41477‐018‐0288‐5

[37]

10.1016/j.addr.2021.05.018

[38]

10.1073/pnas.0910603106

[39]

10.1016/j.cell.2012.11.024

[40]

10.1080/20013078.2017.1286095

[41]

10.1038/s41467-019-12275-6

[42]

10.1016/j.ijpharm.2011.08.017

[43]

10.1016/s0006-3495(97)78282-1

[44]

10.1016/j.jim.2014.06.007

[45]

10.1021/acs.nanolett.1c00094

[46]

10.1038/sj.gt.3300745

[47]

10.1038/mt.2012.180

[48]

10.1261/rna.2255810

[49]

10.1021/acsnano.8b02053

[50]

10.1016/j.addr.2020.06.014

Showing 50 of 75 references

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Details

Published: Oct 01, 2022
Vol/Issue: 11(10)
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Authors

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Jenna McCann