A pH-Mediated Highly Selective System Enabling Simultaneous Analysis of Circulating RNAs Carried by Extracellular Vesicles and Lipoproteins

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.