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miRNA cargo in small extracellular vesicles from mouse primary macrophages and RAW 264.7 cells
Thesis

miRNA cargo in small extracellular vesicles from mouse primary macrophages and RAW 264.7 cells

Jason Tyler DaCunza
Master of Science (M.S.), Drexel University
May 2024
DOI:
https://doi.org/10.17918/00010483
pdf
DaCunza_Jason_20242.44 MB
PDF Embargoed Access, Embargo ends: 30 Jun 2026

Abstract

Chronic pain--Treatment Cell interaction Macrophages miRNA small Extracellular Vesicles (sEVs) Cytology Immunology Inflammation
Chronic pain remains an unmet medical need in the United States due to the limited number of effective treatment options, particularly non-opioid based therapies. Thus, development of new pain treatment paradigms is of critical importance. Small extracellular vesicles (sEVs) are bilipid membrane particles between 30-150 nm in diameter. These particles play an active role in cell-cell communication by transporting a variety of cargo such as nucleic acids, proteins, and lipids. In addition, uptake of sEVs can induce alterations in gene expression and have significant therapeutic potential in a range of diseases. We have previously shown that sEVs from RAW 264.7 macrophages attenuate pain when administered to C57/BL/6 mice. These sEVs were found to contain anti-inflammatory miRNAs that were upregulated when RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS). While these data suggest that macrophage sEVs could potentially be used therapeutically, all prior studies have used sEVs from cell-line macrophages. Hence, it is unknown if the observed in vivo efficacy is specific to RAW 264.7 derived sEVs, or if it applies to macrophage-derived sEVs in general. Additionally, it is unknown if the allogenicity of RAW 264.7 derived sEVs (BLAB/c background) towards C57/BL/6 mice impacts efficacy. This study aims to provide a foundation to determine if rodent primary macrophage derived sEVs are comparable in efficacy to sEVs from RAW 264.7 cells. Primary macrophage cultures of BALB/c and C57BL/6J strains were established and characterized using flow cytometry to confirm differentiation and activation upon LPS treatment. Subsequently, sEVs from LPS-treated and untreated cultures were isolated, characterized, and their miRNAs sequenced. Our initial analysis suggests that primary macrophage sEVs exhibit differences in miRNA signature. Future behavior studies will determine if sEVs from rodent primary macrophages can attenuate pain, which will shed light on the mechanisms governing sEV-mediated pain relief.

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