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Biodistribution of unmodified cardiosphere-derived cell extracellular vesicles using single RNA tracing.

ABSTRACT: Extracellular vesicles (EVs) are potent signalling mediators. Although interest in EV translation is ever-increasing, development efforts are hampered by the inability to reliably assess the uptake of EVs and their RNA cargo. Here, we establish a novel qPCR-based method for the detection of unmodified EVS using an RNA Tracer (DUST). In this proof-of-concept study we use a human-specific Y RNA-derived small RNA (YsRNA) we dub "NT4" that is enriched in cardiosphere-derived cell small EVs (CDC-sEVs). The assay is robust, sensitive, and reproducible. Intravenously administered CDC-sEVs accumulated primarily in the heart on a per mg basis. Cardiac injury enhanced EV uptake in the heart, liver, and brain. Inhibition of EV docking by heparin suppressed uptake variably, while inhibition of endocytosis attenuated uptake in all organs. In vitro, EVs were uptaken more efficiently by macrophages, endothelial cells, and cardiac fibroblasts compared to cardiomyocytes. These findings demonstrate the utility of DUST to assess uptake of EVs in vivo and in vitro.

SUBMITTER: Ciullo A

PROVIDER: S-EPMC8743874 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Biodistribution of unmodified cardiosphere-derived cell extracellular vesicles using single RNA tracing.

Ciullo Alessandra A Li Chang C Li Liang L Ungerleider Korie C KC Peck Kiel K Marbán Eduardo E Ibrahim Ahmed G E AGE

Journal of extracellular vesicles 20220101 1

Extracellular vesicles (EVs) are potent signalling mediators. Although interest in EV translation is ever-increasing, development efforts are hampered by the inability to reliably assess the uptake of EVs and their RNA cargo. Here, we establish a novel qPCR-based method for the detection of unmodified EVS using an RNA Tracer (DUST). In this proof-of-concept study we use a human-specific Y RNA-derived small RNA (YsRNA) we dub "NT4" that is enriched in cardiosphere-derived cell small EVs (CDC-sEVs ...[more]

PMID: 35005847

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Project description:BackgroundMesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are a promising cell-free therapy for acute lung injury (ALI). To date, no studies have investigated their biodistribution in ALI or discerned the timing of administration for maximal lung targeting, which are crucial considerations for clinical translation. Our study aimed to characterize a mouse model of ALI and establish the distribution kinetics and optimal timing of MSC-EV delivery during lung injury.MethodsMSC-EVs were isolated by ultracentrifugation alone (U/C) or tangential flow filtration with ultracentrifugation (TFF-U/C) and characterized by nanoparticle tracking analysis and western blot. A lipopolysaccharide (LPS)-induced mouse model of ALI was established to study the inflammatory response over 72 h. ALI was assessed by histological lung injury score, bronchoalveolar lavage fluid cell count and inflammatory cytokines. For biodistribution studies, ALI mice were intravenously administered fluorescently labeled MSC-EVs to determine the optimal timing of administration and organ-specific biodistribution. Live in vivo and ex vivo fluorescence imaging was conducted at various timepoints post-EV injection.ResultsEVs isolated by either ultracentrifugation alone or TFF-U/C displayed comparable size distribution (~ 50-350 nm) and EV marker expression (CD63/81). TFF-U/C generated a 5.4-fold higher particle concentration and 3.9-fold higher total protein when compared to ultracentrifugation alone. From the inflammatory time-course study, cell count and IL-1β peaked in bronchoalveolar lavage fluid at 24 h after ALI induction. MSC-EVs delivered at 24 h (as opposed to 0.5 h, 5 h or 10 h) after disease induction resulted in a 2.7-4.4-fold higher lung uptake of EVs. Biodistribution studies comparing organ-specific MSC-EV uptake showed progressive lung accumulation up to 48 h post-delivery (threefold higher than the spleen/liver), with a decline at 72 h. Importantly, lung EV fluorescence at 48 h in ALI mice was significantly elevated as compared to control mice. The lung tropism of MSC-EVs was further validated as therapeutically inert EVs derived from HEK293T cells accumulated mainly to the spleen and liver with a 5.5-fold lower distribution to the lungs as compared to MSC-EVs.ConclusionMSC-EVs exhibit maximal lung accumulation when administered during heightened inflammation at 24 h after ALI induction. This lung tropism suggests that MSC-EVs may serve as a practical rescue treatment for acute inflammatory respiratory conditions.

Dataset Information

Biodistribution of unmodified cardiosphere-derived cell extracellular vesicles using single RNA tracing.

Publications

Biodistribution of unmodified cardiosphere-derived cell extracellular vesicles using single RNA tracing.

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