Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour

Abstract

Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named “Sonication and Extrusion‐assisted Active Loading” (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin‐loaded milk‐derived EVs (Dox‐mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10‐fold enhancement of drug encapsulation efficiency compared with passive loading. Single‐particle analysis by nano‐flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox‐mEVs which would otherwise be overlooked by bulk‐based approaches. Correlation analysis between doxorubicin auto‐fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid‐enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid‐probe‐ and immuno‐mediated magnetic isolation techniques to selectively remove the contaminants of non‐lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL‐prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as‐developed cargo‐loading approach and nano‐flow cytometry‐based characterization method will provide an instructive insight in the development of EV‐based delivery systems.

Conscription of Immune Cells by Light‐Activatable Silencing NK‐Derived Exosome (LASNEO) for Synergetic Tumor Eradication

“…[ 15c ] Because both exosome membrane and Ce6 intrinsically were hydrophobic, we tested both co‐incubation and electroporation methods to load Ce6 into SNEO. [ 24 ] It was observed that there was no significant difference between these two methods, and the loading efficiency for incubation saturated at Ce6 concentration at 125 ng µL −1 , with a loading efficiency of ≈13.37% (Figure 2C , and Figure S11 , Supporting Information).…”

Extracellular Vesicles as Novel Drug-Delivery Systems through Intracellular Communications

“…When trying to develop DDSs with EVs, which are natural drug carriers, there are two methods: loading the drug directly into the recovered EVs [121][122][123], and loading drugs into EVs by modifying the function of secretory cells [124][125][126]. In particular, the method of imparting functions to EVs that are extracted by manipulating the cells described above is significantly different from the DDS technology that is applied to conventional artificial nanoparticle preparations.…”

Nutraceuticals in HIV and COVID-19-Related Neurological Complications: Opportunity to Use Extracellular Vesicles as Drug Delivery Modality

“…Naturally occurring EVs attract a large number of researchers across the globe due to numerous advantages over biomaterials in context of a safe and effective drug delivery approach, especially for the therapy of neurodegenerative diseases [ 163 ]. EVs may have sufficient drug loading efficiency and loading capacity compared to other nanoparticles with low/no toxicity [ 146 , 164 ]. Being a natural carrier, EVs can effectively carry biomolecules such as lipids, proteins [ 165 ], and coding and non-coding RNAs that can regulate gene expression in the target cells, and thus provide suitability as an effective drug delivery system for biological molecules [ 138 ].…”