Genetic Engineering of Extracellular Vesicles (EVs) for Targeted Drug Delivery Applications

Introduction: Extracellular vesicles (EVs) have been promoted as promising nanoscale drug delivery systems due to their biocompatibility and ability to cross biological barriers. EVs, however, accumulate in clearance organs such as liver and spleen rather than at desired target sites[1]. Several approaches such as covalent conjugation of targeting ligands to purified EVs, and immobilisation of EVs in targeting biomaterial scaffolds (e.g., hydrogels) [2-4] have been applied to direct EV biodistribution, but these can be difficult to standardise and challenging to scale. Genetic engineering offers a modular and potentially more reproducible control over surface display of targeting proteins on EV. Methods: To establish such a platform, a stable HEK293 producer cell line was generated using a transposon-based PiggyBac system to express a truncated Prostaglandin F2 Receptor Negative Regulator (PTGFRN) fused to the fluorescent protein mCherry as a proof-of-principle model cargo, thereby, evaluating PTGFRN as a scaffold for EV engineering. PTGFRN was selected due to its topology potentially supporting display of targeting ligands and peptides therapeutics towards extracellular targets. Producer cells were imaged to confirm fluorescence and EVs were isolated from conditioned media by size-exclusion chromatography and characterised by cryogenic transmission electron microscopy (cryo-TEM), nanoparticle tracking analysis (NTA), immunoblotting and nanoflow cytometry for mCherry and EV markers. Results and discussion: The engineered HEK293 cell line exhibited strong red fluorescence, indicating stable expression of the PTGFRN–mCherry fusion (Fig 1A). EVs displayed typical vesicular morphology and size distribution by cryo-TEM and NTA (Fig 1B, 1C) and were positive for canonical EV markers (Fig 1D), confirming preservation of EV identity. Single-vesicle nanoflow cytometry demonstrated that ~ 20% of the particles were mCherry-positive and staining with an anti-mCherry antibody, confirmed that mCherry was accessible on the vesicle exterior, validating true surface display (Fig 1E). The mCherry-positive EVs were also positive for CD63 and, to a lesser extent, CD9, indicating that the cellular engineering strategy preserves tetraspanin expression and is consistent with the expected EV heterogeneity of HEK293-derived EVs (Figure 1F, 1G). Conclusion: Using mCherry as a model protein, this PTGFRN-based stable producer cell line provides a genetically encoded traceable platform for EV engineering that can be extended to display targeting ligands. These features position genetic engineering of EVs as a versatile route to design next-generation, targeted EV therapeutics.