Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Area of Science:

• Biotechnology and Nanomedicine
• Drug Delivery Systems
• Molecular Biology

Background:

• Lipid nanoparticles (LNPs) are common drug delivery vesicles but suffer from poor biocompatibility and immune reactions.
• Extracellular vesicles (EVs) offer a promising alternative due to their natural origin, safety, and multifunctionality.
• Efficiently loading large biomolecules like mRNA into EVs remains a significant challenge.

Purpose of the Study:

• To develop a controlled method for loading extracellular vesicles (EVs) with messenger RNA (mRNA).
• To create novel hybrid nanocarriers, termed EV-liposome hybrids (EVLs), for enhanced RNA therapeutics delivery.
• To evaluate the efficiency and scalability of the EVL production and their performance in gene expression.

Main Methods:

• Utilized DNA-mediated, programmed fusion between EVs and mRNA-loaded liposomes.
• Employed real-time microscopy with immobilized EVs for single-particle fusion efficiency analysis.
• Scaled up production using magnetic beads and DNA strand-replacement reactions for EVL collection.

Main Results:

• Demonstrated successful encapsulation of mCherry mRNA within the fused EVL particles.
• Showcased enhanced transfection and improved mRNA translation in HEK293-H cells compared to traditional liposomes or LNPs.
• Achieved a million-fold scale-up in EVL production using magnetic bead-based methods.

Conclusions:

• The developed DNA-mediated fusion technique provides a controlled and efficient method for creating EV-liposome hybrids (EVLs).
• EVLs serve as effective nanocarriers for RNA therapeutics, improving delivery and translation efficacy.
• This approach represents a significant advancement for EV-mediated delivery of RNA therapeutics, overcoming limitations of current synthetic nanoparticles.