Rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes

Abstract

Extracellular vesicles (EVs) can mediate intercellular communication by transferring cargo proteins and nucleic acids between cells. The pathophysiological roles and clinical value of EVs are under intense investigation, yet most studies are limited by technical challenges in the isolation of nanoscale EVs (nEVs). Here, we report a lipid nanoprobe that enables spontaneous labelling and magnetic enrichment of nEVs in 15 minutes, with isolation efficiency and cargo composition similar to what can be achieved by the much slower and bulkier method of ultracentrifugation. We also show that the lipid nanoprobes, which allow for downstream analyses of nucleic acids and proteins, enabled the identification of EGFR and KRAS mutations following nEV isolation from blood plasma from non-small-cell lung-cancer patients. The efficiency and versatility of the lipid nanoprobe opens up opportunities in point-of-care cancer diagnostics.

Figure 1. Schematic of the LNP system for nEV enrichment and downstream analyses

Top and Middle, nEVs from either serum-free cell-culture medium or blood plasma are marked with the labelling probe (top), followed by magnetic separation with the capture probe (middle). Bottom, nEVs and their cargo contents can then be analysed by different methods, such as PCR, Sanger sequencing and NGS sequencing for DNA, RNA staining and RNA NGS for RNA, enzyme-linked immunosorbent assay (ELISA), the bicinchoninic acid assay (BCA) and LC-MS/MS for proteins, and cellular-uptake and wound-healing assays for functionality.

Figure 2. Morphological characterization of the materials and optimization of the LNP system for isolation efficiency

a–d, Morphological characterization by EM. Scale bars, 100 nm. a, SEM image of synthesized MMPs. b,c, TEM (b) and cryo-SEM (c) images showing the morphology of nEVs. d, Cryo-SEM image showing that LP-labelled MDA-MB-231 nEVs (arrowhead) are captured on the surface of the CPs. e–g, Isolation efficiency of MDA-MB-231 nEVs as a function of LP amount (e), incubation time of the LPs with model samples (f), and incubation time of the LPs and CPs (g); Error bars, mean ± s.e.m. (n = 5). h, Isolation efficiency of nEVs from healthy-donor plasma samples as a function of LP amount. Error bars, mean ± s.e.m. (n = 3).

Figure 3. Isolated nEVs provide flexibility in downstream molecular analyses

a, LP-labelled nEVs were enriched on NA-coated well plates followed by RNA-dye staining. Fluorescence intensity increased in direct proportion to total RNA contained in integral nEVs. Error bars, mean ± s.e.m. (n = 4). b, Fluorescently labelled CD9 and EpCAM antibodies were used to detect relevant protein expression of model nEVs released from SK-N-BE(2), MDA-MB-231 and SW620 cells. Error bars, mean ± s.e.m. (n = 20). Top insets show CD9 (green), EpCAM (red) and DAPI (blue) staining of the cells. c, CD63 and GAPDH proteins were extracted and identified from isolated nEVs by western blot. d, DNA and RNA were extracted from isolated nEVs and identified with 2% agarose gel electrophoresis. 1 kbp DNA ladders (labelled as M) indicate the length of fragments. e, Circos plots of nEV DNA of MDA-MB-231 cells isolated by ultracentrifugation (top) and by the LNP (bottom). DNA was sequenced by NGS with 3.3× depth of coverage, mapped to human genome, and plotted with a size window of 100 kbp. Read coverage is expressed in natural logarithmic scale with 0, 3, 6, 9, 12, and 15 reads, from the inside to the outside. f, Pie charts depicting different RNA species and their mapped read-count distributions from MDA-MB-231 nEVs isolated by ultracentrifugation (top) and by the LNP (bottom). Left, full-scale plots; Right, plots zooming into low-abundance RNA species (labelled as ‘others’ in the full-scale plots).

Figure 4. Detection of DNA mutations in nEVs isolated from plasma samples from NSCLC patients

a, Gel electrophoresis of EGFR and KRAS DNA fragments after a first round of PCR amplification (top) and a second round of mutant-enriched nested PCR (bottom). nEVs were isolated from the plasma of patient 24 followed by DNA extraction and PCR amplification. To detect EGFR mutations in exons 19 and 21, first-batch PCR products were enzymatically digested and used as template for the next mutant-enriched nested PCR. M, 1 kbp DNA ladders. b, A KRAS G13D point mutation was detected in the plasma sample of patient 42 by using direct sequencing of traditional PCR products with reverse primers. c, A EGFR L858R point mutation in exon 21 was identified in the plasma sample of patient 28 by sequencing the nested PCR products. d, A deletion mutation in the EGFR exon 19 was identified in the plasma sample of patient 29 by sequencing the nested PCR products. e, Real-time PCR profiles of samples from patients 42 and 51, alongside positive and negative controls. Real-time PCR was performed on all nEV DNA samples to enrich for KRAS mutations. f, Sequencing of the real-time PCR product confirmed the KRAS G13D mutation in the plasma sample of patient 42. g, Sequencing of the real-time PCR product detected a KRAS G12D mutation in the plasma of patient 51.