Extracellular vesicles (EVs) mediate intercellular transport of biomolecular cargo in the body, making them promising delivery vehicles for bioactive compounds. Genetic engineering of producer cells has enabled encapsulation of therapeutic proteins in EVs. However, genetic engineering approaches can be expensive, time-consuming, and incompatible with certain EV sources, such as human plasma and bovine milk. The goal of this study was to develop a quick, versatile, and simple method for loading proteins in EVs post-isolation. Proteins, including CRISPR associated protein 9 (Cas9), were bound to cationic lipids that were further complexed with MDA-MB-231 cell-derived EVs through passive incubation. Size-exclusion chromatography was used to remove components that were not complexed with EVs. The ability of EVs to mediate intracellular delivery of proteins was compared to conventional methods, such as electroporation and commercial protein transfection reagents. The results indicate that EVs retain native features following protein-loading and obtain similar levels of intracellular protein delivery as conventional methods, but display less toxicity. This method opens up opportunities for rapid exploration of EVs for protein delivery.

The variable presence of contaminants in extracellular vesicle (EV) samples is one of the major contributors to a lack of inter‐study reproducibility in the field. Well‐known contaminants include protein aggregates, RNA‐protein complexes and lipoproteins, which resemble EVs in shape, size and/or density. On the contrary, polysaccharides, such as hyaluronic acid (HA), have been overlooked as EV contaminants. Here, it is shown that low and medium molecular weight HA polymers are unexpectedly retained to some extent in EV fractions using two common isolation methods known for high purity: size‐exclusion chromatography and tangential flow filtration. Although these isolation techniques are capable of efficient removal of non‐EV‐associated proteins, this is not the case for HA polymers, which are partially retained in a molecular weight‐dependent manner, especially with size‐exclusion chromatography. The supramolecular structure and hydrodynamic size of HA are likely to contribute to isolation in EV fractions of filtration‐based approaches. Conversely, HA polymers were not retained with ultracentrifugation and polymer‐based precipitation methods, which are known for co‐isolating other types of contaminants. HA has a broad range of immunomodulatory effects, similar to those ascribed to various sources of EVs. Therefore, HA contaminants should be considered in future studies to avoid potential inaccurate attributions of functional effects to EVs.

Extracellular vesicles (EVs) are cell-released nanoparticles that transfer biomolecular content between cells. Among EV-associated biomolecules, microRNAs (miRNAs/miRs) represent one of the most important modulators of signaling pathways in recipient cells. Previous studies have shown that EVs from adipose-derived mesenchymal stromal cells (MSCs) and adipose tissue modulate inflammatory pathways in macrophages. In this study, the effects of miRNAs that are abundant in adipose tissue EVs and other biogenic nanoparticles (BiNPs) were assessed in terms of altering Toll-like receptor 4 (TLR4)-induced cytokines. TLR-4 signaling in macrophages is often triggered by pathogen or damage-induced inflammation and is associated with several diseases. This study demonstrates that miR-451a, which is abundant in adipose tissue BiNPs, suppresses pro-inflammatory cytokines and increases anti-inflammatory cytokines associated with the TLR4 pathway. Therefore, miR-451a may be partially responsible for immunomodulatory effects of adipose tissue-derived BiNPs.

In vitro cell-based characterization methods of nanoparticles are generally static and require the use of secondary analysis techniques and labeling agents. In this study, bare niosomes and chitosan-coated niosomes (chitosomes) and their interactions with intestinal cells are studied under dynamic conditions and without fluorescent probes, using surface plasmon resonance (SPR)-based cell sensing. Niosomes and chitosomes were synthesized by using Tween 20 and cholesterol in a 15 mM:15 mM ratio and then characterized by dynamic light scattering (DLS). DLS analysis demonstrated that bare niosomes had average sizes of ∼125 nm, polydispersity index (PDI) below 0.2, and a negative zeta (ζ)-potential of -35.6 mV. In turn, chitosomes had increased sizes up to ∼180 nm, with a PDI of 0.2-0.3 and a highly positive ζ-potential of +57.9 mV. The viability of HT29-MTX, Caco-2, and Caco-2/HT29-MTX cocultured cells showed that both niosomes and chitosomes are cytocompatible up to concentrations of 31.6 μg/mL for at least 240 min. SPR analysis demonstrated that chitosomes interact more efficiently with HT29-MTX, Caco-2, and Caco-2/HT29-MTX cocultures compared to bare niosomes. The resulting SPR measurements were further supported by confocal microscopy and flow cytometry studies, which demonstrated that this method is a useful complementary or even alternative tool to directly characterize the interactions between niosomes and in vitro cell models in label-free and real-time conditions.

Extracellular vesicles (EVs) are biological nanoparticles that play important roles in (patho)physiological processes and are promising new therapeutic and diagnostic tools. Recent evidence suggests that other circulating biological nanoparticles, primarily lipoproteins, bind to EVs, changing their biological identity. Such binding has been demonstrated with complex qualitative techniques, such as cryogenic transmission electron microscopy. There is a need to rapidly and simply quantify EV‐lipoprotein binding, as such complexes could have major implications for EV biology and medical applications. This study developed lipoprotein association fluorometry (LAF; based on fluorescent lipophilic indocarbocyanine dyes), as a first‐of‐its‐kind, simple and quick assay to assess EV binding to lipoproteins. The LAF assay was validated with synthetic nanoparticles, small molecules, polymers and proteins that display known interactions with lipoproteins. The LAF assay demonstrates that EVs from various human and non‐human (nematode and bacteria) sources bind to very‐low‐density lipoprotein (VLDL) and low‐density lipoprotein (LDL). Notably, EVs derived from cancerous cells displayed substantially increased binding to VLDL, LDL and plasma compared to EVs from normal cells. Additionally, the LAF assay revealed that EVs from metastatic cancer cells bound to VLDL to a greater extent than those from corresponding patient‐matched non‐metastatic cancer cells. On the contrary, EVs displayed minimal binding to high‐density lipoprotein (HDL). Taken together, the LAF assay is capable of measuring EV‐lipoprotein binding in a simple, rapid and semi‐quantitative manner, leading to new opportunities to probe EV biology and develop novel therapeutics, and diagnostics.