Unraveling the Multifaceted Roles of Extracellular Vesicles: Insights into Biology, Pharmacology, and Pharmaceutical Applications for Drug Delivery.

Extracellular vesicles (EVs) are nanoparticles released from various cell types that have emerged as powerful new therapeutic option for a variety of diseases. EVs are involved in the transmission of biological signals between cells and in the regulation of a variety of biological processes, highlighting them as potential novel targets/platforms for therapeutics intervention and/or delivery. Therefore, it is necessary to investigate new aspects of EVs' biogenesis, biodistribution, metabolism, and excretion as well as safety/compatibility of both unmodified and engineered EVs upon administration in different pharmaceutical dosage forms and delivery systems. In this review, we summarize the current knowledge of essential physiological and pathological roles of EVs in different organs and organ systems. We provide an overview regarding application of EVs as therapeutic targets, therapeutics, and drug delivery platforms. We also explore various approaches implemented over the years to improve the dosage of specific EV products for different administration routes.

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell-extracellular vesicle preparations.

BACKGROUND AIMS: Extracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed. METHODS: By combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia). RESULTS: The authors' analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types. CONCLUSIONS: Overall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors' MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.

Liposomes or Extracellular Vesicles: A Comprehensive Comparison of Both Lipid Bilayer Vesicles for Pulmonary Drug Delivery.

The rapid and non-invasive pulmonary drug delivery (PDD) has attracted great attention compared to the other routes. However, nanoparticle platforms, like liposomes (LPs) and extracellular vesicles (EVs), require extensive reformulation to suit the requirements of PDD. LPs are artificial vesicles composed of lipid bilayers capable of encapsulating hydrophilic and hydrophobic substances, whereas EVs are natural vesicles secreted by cells. Additionally, novel LPs-EVs hybrid vesicles may confer the best of both. The preparation methods of EVs are distinguished from LPs since they rely mainly on extraction and purification, whereas the LPs are synthesized from their basic ingredients. Similarly, drug loading methods into/onto EVs are distinguished whereby they are cell- or non-cell-based, whereas LPs are loaded via passive or active approaches. This review discusses the progress in LPs and EVs as well as hybrid vesicles with a special focus on PDD. It also provides a perspective comparison between LPs and EVs from various aspects (composition, preparation/extraction, drug loading, and large-scale manufacturing) as well as the future prospects for inhaled therapeutics. In addition, it discusses the challenges that may be encountered in scaling up the production and presents our view regarding the clinical translation of the laboratory findings into commercial products.