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  • Title: A pH-Mediated Highly Selective System Enabling Simultaneous Analysis of Circulating RNAs Carried by Extracellular Vesicles and Lipoproteins.
    Author: Yang R, Lou D, Xia K, Sun L, Zhu Q.
    Journal: Anal Chem; 2023 Dec 26; 95(51):18803-18813. PubMed ID: 38078945.
    Abstract:
    Extracellular vesicles (EVs) and lipoproteins (LPPs) serve as important carriers of circulating miRNAs in peripheral blood, offering immense potential for disease diagnosis and therapeutic interventions. Due to their shared physicochemical attributes, EVs and LPPs are frequently coisolated, potentially leading to misunderstandings regarding their distinct functional roles in physiological and pathological processes. Here, we report a highly selective magnetic system based on the pH-mediated affinity displayed by cibacron blue (CB) toward EVs and LPPs, enabling successful separation and collection of these two nanoparticles without cross-contamination for subsequent circulating RNA analysis. First, we found that CB-modified magnetic beads (CBMBs) exhibit a strong affinity toward LPP particles while displaying little interaction with EVs in standard samples under physiological pH conditions. We further demonstrate that the affinity between CB molecules and bionanoparticles in plasma samples is highly pH-dependent. Specifically, CBMBs show affinities for both LPP and EV particles under neutral and acidic conditions. However, at basic pH levels, CB molecules selectively bind only to LPP particles. Consequently, the remaining EV particles present in plasma are subsequently isolated by using titanium dioxide-modified beads (TiMBs) through phospholipid affinity. The simultaneous analysis of the transcriptomic contents of EV and LPP reveals clear differences in their small RNA profiles, with the differentially expressed RNAs reflecting distinct biological processes. Significantly, in a proof-of-concept study, we successfully demonstrated a strong correlation between miRNAs carried by both EV and LPP particles with the occurrence of ocular neovascularization during the progression of diabetic retinopathy. The involved miRNAs may serve as potential biomarkers for DR diagnostics and severity classification. To sum up, this pH-mediated separation system is not only user-friendly but also highly compatible, rendering it a potent tool for probing the molecular compositions, biomarkers, and underlying biological mechanisms of EVs and LPPs.
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