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Abstract Extracellular vesicles (EVs) have shown promise as potential therapeutics for the treatment of various diseases. However, their rapid clearance after administration could be a limitation in certain therapeutic settings. To solve this, an engineering strategy is employed to decorate albumin onto the surface of the EVs through surface display of albumin binding domains (ABDs). ABDs were either included in the extracellular loops of select EV‐enriched tetraspanins (CD63, CD9 and CD81) or directly fused to the extracellular terminal of single transmembrane EV‐sorting domains, such as Lamp2B. These engineered EVs exert robust binding capacity to human serum albumins (HSA) in vitro and mouse serum albumins (MSA) after injection in mice. By binding to MSA, circulating time of EVs dramatically increases after different routes of injection in different strains of mice. Moreover, these engineered EVs show considerable lymph node (LN) and solid tumour accumulation, which can be utilized when using EVs for immunomodulation, cancer‐ and/or immunotherapy. The increased circulation time of EVs may also be important when combined with tissue‐specific targeting ligands and could provide significant benefit for their therapeutic use in a variety of disease indications.

Abstract Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS‐HAT). We report that PBS‐HAT buffer facilitates clearly improved short‐term and long‐term EV preservation for samples stored at ‐80°C, stability throughout several freeze‐thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

The clinical use of chemotherapeutics is limited by several factors, including low cellular uptake, short circulation time, and severe adverse effects. Extracellular vesicles (EVs) have been suggested as a drug delivery platform with the potential to overcome these limitations. EVs are cell-derived, lipid bilayer nanoparticles, important for intercellular communication. They can transport bioactive cargo throughout the body, surmount biological barriers, and target a variety of tissues. Several small molecule drugs have been successfully incorporated into the lumen of EVs, permitting efficient transport to tumour tissue, increasing therapeutic potency, and reducing adverse effects. However, the cargo loading is often inadequate and refined methods are a prerequisite for successful utilisation of the platform. By systematically evaluating the effect of altered loading parameters for electroporation, such as total number of EVs, drug to EV ratio, buffers, pulse capacitance, and field strength, we were able to distinguish tendencies and correlations. This allowed us to design an optimised electroporation protocol for loading EVs with the chemotherapeutic drug doxorubicin. The loading technique demonstrated improved cargo loading and EV recovery, as well as drug potency, with a 190-fold increased response compared to naked doxorubicin.