RNA interference opened new approaches for disease treatment but safe and efficient cell delivery remains a bottleneck. Extracellular vesicles (EVs) are known to naturally shuttle RNA. Due to their potent cell internalization and low‐cost scalability, milk‐derived EVs in particular are considered promising RNA delivery systems. However, low drug loading currently impedes their use. Here, innovative exogenous loading strategies for small interfering RNA (siRNA) are explored and systematically compared regarding encapsulation efficiency, loading capacity, and loading concentration. Firstly, siRNA is pre‐accumulated in liposomes or stabilized calcium phosphate nanoparticles (CaP‐NP). The selected systems, which exhibited neutral or negative zeta potentials, are then applied for EV loading. Secondly, EVs are concentrated and applied to protocols known for liposome loading: dehydration‐rehydration of vesicles, based on freeze‐drying, and mixing by dual asymmetric centrifugation (DAC) after ultracentrifugation. Additionally, DAC after EV ultracentrifugation is combined with CaP‐NP leading to a synergistic loading performance. The balance between energy input for siRNA loading and EV integrity is evaluated by monitoring the EV size, marker proteins, and morphology. For the EV‐based siRNA formulation via DAC plus CaP‐NP, EV properties are sufficiently maintained to protect the siRNA from degradation and deliver cell‐death siRNA dose‐dependently in Caco‐2 cells.

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Extracellular vesicles (EVs) are widely recognized for their potential as drug delivery systems. EVs are membranous nanoparticles shed from cells. Among their natural features are their ability to shield cargo molecules against degradation and enable their functional internalization into target cells. Especially biological or bio‐inspired large molecules (LMs), like nucleic acids, proteins, peptides, and others, may profit from encapsulation in EVs for drug delivery purposes. In the last years, a variety of loading protocols are explored for different LMs. The lack of standardization in the EV drug delivery field has impeded their comparability so far. Currently, the first reporting frameworks and workflows for EV drug loading are proposed. The aim of this review is to summarize these evolving standardization approaches and set recently developed methods into context. This will allow for enhanced comparability of future work on EV drug loading with LMs.