Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions

Extracellular vesicles (EVs), naturally occurring lipid nanoparticles, are gaining traction as promising tools for delivering therapeutic molecules. However, efficiently loading specific proteins into EVs for targeted delivery has posed a significant challenge—until now.

Innovative Protein Engineering: Recent work by researchers at Northwestern University has unveiled a breakthrough in the field by strategically designing proteins to traffic to the cell’s plasma membrane and associate with lipid rafts. This approach enhances the loading of diverse transmembrane and peripheral membrane proteins into EVs, paving the way for precise delivery of therapeutic cargo.

Protein Tagging for Enhanced Loading: Researchers have identified lipid tags capable of significantly increasing the loading capacity of EVs. These tags facilitate the efficient packaging and functional delivery of engineered transcription factors—a critical step toward modulating gene expression in targeted cells.

EV membrane physical properties and protein content mirror those of lipid rafts

Fig. 1

a Schematic of our hypothesis that lipid raft association could be used as a handle to load proteins into EVs. b Overall analysis and experimental workflow. We used RaftProt 2.0 and Exocarta to understand features of proteins found in EVs and lipid rafts (I); we then built a library of structurally diverse proteins to understand how such features affect protein trafficking, interactions with lipid rafts, and loading into EVs (II); applying these design rules, we demonstrate how lipid-protein interactions can be used to functionally deliver cargo to cells via EVs (III). c Laurdan generalized polarization (GP) of ordered liposomes (LO) composed of 70 mol% DPPC/30 mol% Chol, disordered liposomes (LD) composed of 70 mol% DOPC/30 mol% Chol, HEK293FT cells, giant plasma membrane vesicles (GPMVs) and vesicles from the high-speed centrifugation EV fraction (HS-EVs) and ultracentrifugation EV fraction (UC-EVs) derived from HEK293FT cells. HS-EV and UC-EVs had high Laurdan GP, similar to LO membranes as calculated by Eq. 1. Each dot represents an independent experiment (n ≥ 3). A one-way ANOVA was performed to compare the Laurdan GP of HS-EVs and UC-EVs to the Laurdan GP of all other membranes measured, and comparisons were evaluated using the Sidak multiple comparisons correction. Both EV populations were significantly different from all other vesicle populations except one another (****p < 0.0001). d The frequency at which human membrane-associated proteins and raft proteins are found in EVs as calculated via bioinformatic analysis. Raft associated proteins are more frequently found in EVs compared to a random selection of human membrane-associated proteins. Each dot represents a separate query of 100 human proteins (n = 3). An unpaired, two-tailed t-test was performed to compare the fraction of all proteins and raft associated proteins found in EVs. The fraction of raft associated proteins found in EVs was significantly different from the fraction of all proteins (p < 0.0001). n ≥ 3, error bars represent the standard error of the mean (SEM) throughout the figure. 

Towards Future Therapeutics: The implications of this technology are profound. By leveraging these advancements, scientists envision developing novel EV-based therapeutics capable of delivering a wide range of macromolecular cargo—from proteins to nucleic acids—across biological barriers for therapeutic benefit.

Promise for Personalized Medicine: This breakthrough not only enhances our ability to load specific proteins into EVs but also opens avenues for personalized medicine. Tailored EVs loaded with precise therapeutic payloads could revolutionize treatment strategies for a variety of diseases, including cancers and genetic disorders.

Conclusion: This innovation in engineering EVs to efficiently load and deliver therapeutic proteins marks a significant leap forward in biotechnology. As research continues to unravel the full potential of EV-based therapeutics, the future holds promise for transformative treatments that harness the natural capabilities of these tiny lipid nanoparticles.

Future Directions: Looking ahead, further research will focus on refining the engineering processes, expanding cargo options, and advancing clinical applications. This journey promises to bring us closer to a new era of targeted, effective, and minimally invasive therapies using EVs as delivery vehicles.

This research underscores the transformative potential of EVs in medicine, offering hope for more effective treatments that harness the body’s own natural delivery systems to combat disease.

Peruzzi JA, Gunnels TF, Edelstein HI, Lu P, Baker D, Leonard JN, Kamat NP. (2024) Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions. Nat Commun 15(1):5618. [article]

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