DNAJB6-Containing Extracellular Vesicles as Chaperone Delivery Systems: A Proteomic Analysis.

Cell-derived extracellular vesicles (EVs) are effectors of cell-to-cell communication that are in the spotlight as promising candidates for in vivo drug delivery because of their ability to enter cells and deliver cargo. For example, proteins of interest can be loaded into EVs to mediate protein transfer into target cells. To determine causality between EV content and function, which is also important to assess the clinical safety of EVs, it is crucial to comprehensively characterize their complete molecular composition. Here, we investigated EVs loaded with the chaperone protein DNAJB6. Chaperone proteins assist in protein folding and have been suggested to alleviate protein aggregation diseases, such as Alzheimer's disease and Huntington's disease. We analyzed and compared the proteome of EVs isolated from wildtype HEK293T cells with that of EVs from HEK 293T cells overexpressing DNAJB6-WT or loss-of-function mutant DNAJB6-M3. Comprehensive analysis of proteomics data showed enhanced levels of DNAJB6 as well as protein-folding-related proteins in EVs derived from DNAJB6-overexpression cells. Interestingly, upregulation of a chaperone and its protein-folding-related proteins resulted in downregulation of another chaperone plus its related proteins, and vice versa. This implies the presence of compensatory mechanisms in the cellular expression of chaperones. Collectively, we provide the proteomic EV signatures underlying EV mediated DNAJB6 transmission by HEK293T cells, with the aim of establishing a causal relationship between EV protein content and EV function.

Macrophages Release Extracellular Vesicles of Different Properties and Composition Following Exposure to Nanoparticles.

Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication.

Time- and Space-Resolved Flow-Cytometry of Cell Organelles to Quantify Nanoparticle Uptake and Intracellular Trafficking by Cells.

The design of targeted nanomedicines requires intracellular space- and time-resolved data of nanoparticle distribution following uptake. Current methods to study intracellular trafficking, such as dynamic colocalization by fluorescence microscopy in live cells, are usually low throughput and require extensive analysis of large datasets to quantify colocalization in several individual cells. Here a method based on flow cytometry to easily detect and characterize the organelles in which nanoparticles are internalized and trafficked over time is proposed. Conventional cell fractionation methods are combined with immunostaining and high-sensitivity organelle flow cytometry to get space-resolved data of nanoparticle intracellular distribution. By extracting the organelles at different times, time-resolved data of nanoparticle intracellular trafficking are obtained. The method is validated by determining how nanoparticle size affects the kinetics of arrival to the lysosomes. The results demonstrate that this method allows high-throughput analysis of nanoparticle uptake and intracellular trafficking by cells, therefore it can be used to determine how nanoparticle design affects their intracellular behavior.