Exo-Fect™ Exosome Transfection

Exo-Fect is a novel nucleic acid transfer agent that enables the transfection of nucleic acids directly into isolated exosomes. The transfected siRNA, microRNA, mRNA or even plasmid DNA can then be shuttled into target cells via the transfected exosome vesicles. Simply combine isolated exosomes with Exo-Fect and the nucleic acid of your choice to generate exosome delivery vehicles. The protocol takes less than an hour and is highly efficient at placing nucleic acids into exosomes for transport. All Exo-Fect kits come with a Texas-Red labeled positive control, non-targeting siRNA to confirm exosome transfections. The term “FedExosomes” was coined by Dr. Michelle E. Marcus and Dr. Joshua N. Leonard in their article, “FedExosomes: Engineering Therapeutic Biological Nanoparticles that Truly Deliver”published in Pharmaceuticals (Basel), May 2013; 6(5): 659-680.

SIMPLE, FAST PROTOCOL

• No electroporation required
• Load siRNA and microRNA
• Works with mRNA as well
• Transfer plasmid DNA into exosomes
• Insert small molecule drugs into exosomes

siRNA & microRNA

A Texas-Red end-labeled siRNA (20 pmol) was transfected into exosomes using Exo-Fect. The far left panels show the transfection of the fluorescently labeled siRNA into exosomes that were then immunocaptured on CD63 magnetic beads. The right panels show the transfection of unlabeled siRNA into exosomes that were then immunocaptured on CD63 magnetic beads. The magnetic beads were imaged after overnight capture of transfected HEK293 exosomes on CD63 immunobeads (catalog# EXOFLOW32A-CD63).

The transfected, CD63-captured exosomes were eluted from the beads and then added to HEK293 cells grown in exosome-depleted media (catalog# EXOFBS-50A-1). The transfected exosomes were allowed to dock and interact with the cells for 1-2 days and then the cells were imaged to monitor siRNA cargo delivery.

mRNA & Plasmid DNA

ExoFect kits work with longer RNAs, like mRNA. We transfected 1 ug of an mRNA encoding RFP into exosomes. These exosomes were then added to HEK293 cells and imaged for RFP protein production after 24 hours. Exo-Fect also enables the transfection of plasmid DNA into exosomes. We used 5 ug of plasmid DNA encoding a GFP gene, transfected exosomes and then added to HEK293 cells. The cells were imaged for GFP protein presence after 48 hours. The image data are shown below.

Small Molecule Drugs

Small moleculae drugs can also be Exo-Fected into exosomes for delivery to target cells. SBI’s cumate system features a cumate switch promoter that is highly suppressed by its cognate CymR repressor protein abd can switch on with the addition of the small moleucle inducer, cumate. We tested the loading of cumate into exosomes using the Exo-Fect system and then added those exosomes to cells containing a cumate switch GFP reporter construct. The control exosomes were untransfected exosomes
and the cumate-transfected exosomes were treated with Exo-Fect and 450 ug cumate solution. The experimental approach and reporter HEK293 cell data are shown below. The Exo-Fect technology is able to successfully load small molecule drugs into exosomes that are then able to deliver the drug to target cells.

Online Resources

TECHNICAL REFERENCES AND PRODUCT CITATIONS

• Marcus ME, Leonard JN. FedExosomes: Engineering Therapeutic Biological Nanoparticles that Truly Deliver. Pharmaceuticals (Basel). 2013;6(5):659-80.
• Zomer A, Vendrig T, Hopmans ES, van Eijndhoven M, Middeldorp JM, Pegtel DM. Exosomes: Fit to deliver small RNA. Commun Integr Biol. 2010 Sep;3(5):447-50.
• Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM. Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6328-33.
• Momen-Heravi F, Bala S, Bukong T, Szabo G. Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages. Nanomedicine. 2014 Mar 29. pii: S1549-9634(14)00132-4.

Products

Exo-Fect Exosome Transfection Kits

ExoQuick and ExoQuick-TC Products

The ExoQuick serum reaction size is based on using 250 µl serum for exosome isolation and ExoQuick-LP is based on a 100 µl starting serum sample.

The ExoQuick-TC reagent provides enough for either 10 reactions (EXOTC10A-1) or 50 reactions (EXOTC50A-1) from 5 ml Media or Urine for exosome isolation.

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Exo-Flow™: Purify Exosomes using Immunoaffinity beads – Selectively capture exosomes on magnetic beads for flow sorting to quantify and purify distinct subpopulations of exosomes.

Exo-FBS exosome-depleted FBS media supplement – Study exosomes from cultured cells and not from fetal bovine serum.

ExoAB and ExoELISA™ Kits – Detect and quantitate exosomes with CD63, CD9, CD81 and Hsp70 antibodies.

SeraMir™ Exosome RNA Amplification Kit – Isolate and convert exosomal RNAs including microRNAs into cDNA for qPCR profiling, Microarrays and  NextGen Sequencing applications.
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Purified Human and Mouse Exosomes