MiRNA Extraction from Cell-Free Biofluid Using Protein Corona Formed around Carboxyl Magnetic Nanoparticles

Abstract

MicroRNA (miRNA) in urine has been considered as a potential biomarker for early-stage diagnosis of multiple diseases like urinary system cancer, kidney injury and diabetes, owing to their many demonstrated advantages including long-term stability and noninvasiveness. However, the traditional enrichment and extraction processes of miRNAs from urine are cumbersome and tedious due to the low concentration and multiple carriers of miRNAs. Herein, we present a novel method to collect low concentrations of miRNAs from dilute solutions such as urine and cell culture medium. 10-nm core sized magnetic nanoparticles with carboxylic acid coating can adsorb low-concentration proteins, and form protein corona which makes them easy to aggregate and precipitate for subsequent isolation. In urine and cell culture medium, these nanoparticles can aggregate with proteins, including miRNAs-associated protein Argonaute 2 and microvesicle-related proteins, to form precipitates, so that miRNAs can be easily extracted from pellets by small amount of lysis buffer for subsequent analysis such as real-time PCR. Our method provides a facile way to enrich miRNAs from biofluids without the need of ultracentrifugation and immunoprecipitations, bringing remarkable convenience to miRNAs-based biomarker research.

Isolation and Quantification of miRNA from the Biomolecular Corona on Mesoporous Silica Nanoparticles

“…The results ( Figure 4 A) indicate that in all cases, the presence of proteins promotes a significant increase of miRNA adsorption on MSN-NH 2 (AII, p < 0.01; C3c, p < 0.05; AII + C3c, p < 0.05), independently of the protein size. This was formerly pointed out [ 30 ], and it is in agreement with the results of Xu et al [ 25 ] who analyzed miRNA from the corona of magnetic nanoparticles after interaction with urine samples. These authors reported that miRNAs can associate with specific proteins of the corona, which promotes their clearance from the liquid medium.…”

“…To our knowledge, no previous investigation on the analysis of miRNA from blood biomolecular corona has been carried out. Indeed, the only paper exploring its diagnostic possibilities refers to miRNA determination by using magnetic nanoparticles (MNPs) functionalized with carboxylic acids to collect and enrich proteins in urine and cell culture media [ 25 ]. That work points out that miRNAs are frequently associated with specific serum protein complexes for protection from digestion by RNases.…”

The biomolecule corona of lipid nanoparticles contains circulating cell-free DNA

“…Recently, Qian and colleagues investigated the interaction of negatively-charged, carboxylated magnetic NPs with miRNAs in rat urine samples and suggested an RNA-induced silencing complex (RISC)-mediated adsorption of miRNA. 33 In a different study, Yun et al developed a planarsubstrate based protocol in order to investigate the interaction of metal-phenolic network (MPN)-based nanomaterials with multiple biomolecules including synthetic single stranded 40 bp DNA fragments. 31 It is important to note however, that these DNA molecules do not reflect the size and structure of endogenous plasma cfDNA, nor the molecular complexity of blood.…”

“…This could be partially due to the experimental challenges in identifying minute levels of blood-circulating nucleic acids on the NPs surfaces. The very few studies that have tried to investigate any role that nucleic acids may play in the biomolecule corona, focused on miRNAs using rat urine samples, 33 or attempted to understand the interaction of NPs with added synthetic DNA fragments in solution. 31,34 NPs have also been extensively used as biosensors or as nano-carriers to deliver nucleic acids, with positively charged lipid-, polymeric-, gold-and carbon-based (among many other) NPs used to deliver electrostaticallycomplexed nucleic acids for therapeutic applications.…”

Extracellular Vesicles: New Endogenous Shuttles for miRNAs in Cancer Diagnosis and Therapy?

“…In the author's opinion, this method provides an easy way to enrich miRNAs from biofluids without the need of ultracentrifugation and immunoprecipitations, bringing remarkable convenience to miRNAs-based biomarker research. [106].…”