Cargo loading is the process of introducing functional molecules into extracellular vesicles (EVs) to enable targeted delivery and therapeutic action.

• Proteins (e.g., Cas9, Cre recombinase)
• Nucleic acids (e.g., mRNA, siRNA)
• Small molecules and drugs
• Fluorescent tags (e.g., EGFP, mCherry)

• 'Passive loading': relies on natural EV sorting (low efficiency)
• 'Engineered loading': uses molecular tags to actively recruit cargo during EV biogenesis
• 'Post-isolation loading': includes electroporation, sonication, or incubation methods

In Obuchi et al. (2025), CD63 was modified to recruit nanobody-tagged cargo via mCherry interaction, enabling selective and efficient loading into EVs.