Engineered small extracellular vesicles as a versatile platform to efficiently load ferulic acid via an esterase-responsive active loading strategy

As nano-drug carriers, small extracellular vesicles (sEVs) have shown unique advantages, but their drug loading and encapsulation efficiency are far from being satisfied, especially for the loading of hydrophilic small-molecule drugs. Inspired by the strategies of active loading of liposomal nanomedicines, pre-drug design and immobilization enzyme, here we developed a new platform, named Esterase-responsive Active Loading (EAL), for the efficient and stable drug encapsulation of sEVs. Widely used ferulic acid ester derivatives were chosen as prodrugs based on the EAL of engineered sEVs to establish a continuous transmembrane ion gradient for achieving efficient loading of active molecule ferulic acid into sEVs. The EAL showed that the drug loading and encapsulation efficiency were around 6-fold and 5-fold higher than passive loading, respectively. Moreover, characterization by nano-flow cytometry and Malvern particle size analyzer showed that differential ultracentrifugation combined with multiple types of membrane filtration methods can achieve large-scale and high-quality production of sEVs. Finally, extracellular and intracellular assessments further confirmed the superior performance of the EAL-prepared sEVs-loaded ferulic acid preparation in terms of slow release and low toxicity. Taken together, these findings will provide an instructive insight into the development of sEV- based delivery systems.

Automated summary

In this study, a new platform named Esterase-responsive Active Loading (EAL) was developed for efficient and stable drug encapsulation of small extracellular vesicles (sEVs). The EAL utilized engineered sEVs to establish a continuous transmembrane ion gradient for active loading of the drug ferulic acid. The results showed that the EAL significantly improved drug loading and encapsulation efficiency compared to passive loading. Furthermore, extracellular and intracellular assessments confirmed the slow release and low toxicity of the EAL-prepared sEVs-loaded ferulic acid preparation.