Extracellular Vesicle and Lipoprotein Interactions.

Extracellular vesicles and lipoproteins are lipid-based biological nanoparticles that play important roles in (patho)physiology. Recent evidence suggests that extracellular vesicles and lipoproteins can interact to form functional complexes. Such complexes have been observed in biofluids from healthy human donors and in various in vitro disease models such as breast cancer and hepatitis C infection. Lipoprotein components can also form part of the biomolecular corona that surrounds extracellular vesicles and contributes to biological identity. Potential mechanisms and the functional relevance of extracellular vesicle-lipoprotein complexes remain poorly understood. This Review addresses the current knowledge of the extracellular vesicle-lipoprotein interface while drawing on pre-existing knowledge of liposome interactions with biological nanoparticles. There is an urgent need for further research on the lipoprotein-extracellular vesicle interface, which could return important mechanistic, therapeutic, and diagnostic findings.

Glycan Node Analysis Detects Varying Glycosaminoglycan Levels in Melanoma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

High-throughput analysis of glycan sorting into extracellular vesicles.

Extracellular vesicles (EVs) are cell-released vesicles that mediate intercellular communication by transferring bioactive cargo. Protein and RNA sorting into EVs has been extensively assessed, while selective enrichment of glycans in EVs remains less explored. In this study, a mass spectrometry-based approach, glycan node analysis (GNA), was applied to broadly assess the sorting of glycan features into EVs. Two metastatic variants (lung and bone) generated in mouse modes from the MDA-MB-231 human breast cancer cell line were assessed, as these EVs are known to contain distinct organotropic biomolecules. EVs were isolated from conditioned cell culture medium by tangential flow filtration and authenticated by standard techniques. GNA analysis revealed selective enrichment of several glycan features in EVs compared to the originating cells, particularly those associated with binding to the extracellular matrix, which was also observed in EVs from the parental MDA-MB-231 cell line (human pleural metastases). The bone-tropic variant displayed enrichment of distinct EV glycan features compared to the lung-tropic one. Additionally, the metastatic variants generated in mouse models displayed reduced EV glycan sorting compared to the parental metastatic cell line. This study represents the first comprehensive assessment of differences in glycan features between EVs and originating cells and provides evidence that the diversity of EV glycan sorting is reduced upon generation of variant cell lines in mouse models. Future research is likely to uncover novel mechanisms of EV glycan sorting, shed light on glycan features for EV authentication or biomarker purposes, and assess functional roles of the EV glycocode in (patho)physiology.

Beyond gold nanoparticles cytotoxicity: Potential to impair metastasis hallmarks.

Gold nanoparticle (AuNP)-based systems have been extensively investigated as diagnostic and therapeutic agents due to their tunable properties and easy surface functionalization. Upon cell uptake, AuNPs present an inherent cell impairment potential based on organelle and macromolecules damage, leading to cell death. Such cytotoxicity is concentration-dependent and completely undesirable, especially if unspecific. However, under non-cytotoxic concentrations, internalized AuNPs could potentially weaken cells and act as antitumor agents. Therefore, this study aimed to investigate the antitumor effect of ultrasmall AuNPs (~3 nm) stabilized by the anionic polysaccharide gum arabic (GA-AuNPs). Other than intrinsic cytotoxicity, the focus was downregulation of cancer hallmarks of aggressive tumors, using a highly metastatic model of melanoma. We first demonstrated that GA-AuNPs showed excellent stability under biological environment. Non-cytotoxic concentrations to seven different cell lines, including tumorigenic and non-tumorigenic cells, were determined by standard 2D in vitro assays. Gold concentrations ≤ 2.4 mg L-1 (16.5 nM AuNPs) were non-cytotoxic and therefore chosen for further analyses. Cells exposed to GA-AuNPs were uptaken by melanoma cells through endocytic processes. Next we described remarkable biological properties using non-cytotoxic concentrations of this nanomaterial. Invasion through an extracellular matrix barrier as well as 3D growth capacity (anchorage-independent colony formation and spheroids growth) were negatively affected by 2.4 mg L-1 GA-AuNPs. Additionally, exposed spheroids showed morphological changes, suggesting that GA-AuNPs could penetrate into the preformed tumor and affect its integrity. All together these results demonstrate that side effects, such as cytotoxicity, can be avoided by choosing the right concentration, nevertheless, preserving desirable effects such as modulation of key tumor cell malignancy features.