Rapid separation of blood plasma exosomes from low-density lipoproteins via a hydrophobic interaction chromatography method on a polyester capillary-channeled polymer fiber phase

Abstract

Exosomes are membrane-bound, cell-secreted vesicles, with sizes ranging from 30 to 150 nm. Exosomes in blood plasma have become proposed targets as measurable indicators of disease conditions. Current methods for plasma-based exosome isolation are time-consuming, complex, and have high operational costs. One of the most commonly reported shortcomings of current isolation protocols is the co-extraction of lipoproteins (e.g. low-density lipoproteins, LDLs) with the target exosomes. This report describes the use of a rapid, single-operation hydrophobic interaction chromatography (HIC) procedure on a polyester (PET) capillary-channeled polymer (C-CP) fiber column, demonstrating the ability to efficiently purify exosomes. The method has previously been demonstrated for isolation of exosomes from diverse biological matrices, but questions were raised about the potential co-elution of LDLs. In the method described herein, a step-gradient procedure sequentially elutes spiked lipoproteins and blood plasma-originating exosomes in 10 min, with the LDLs excluded from the desired exosome fraction. Mass spectrometry (MS) was used to characterize an impurity in the primary LDL material, identifying the presence of exosomal material. Transmission electron microscopy (TEM) and an enzyme-linked immunosorbent assay (ELISA) were used to identify the various elution components. The method serves both as a rapid means of high purity exosome isolation as well as a screening tool for the purity of LDL samples with respect to extracellular vesicles.

Keywords: Capillary-channeled polymer (C-CP); Enzyme-linked immunosorbent assay (ELISA); Exosomes; Extracellular vesicles (EVs); Fibers; Hydrophobic interaction chromatography; Low-density lipoprotein (LDL); Proteomics; Transmission electron microscopy (TEM).

Figure 1.

Diagrammatic representation of the experimental apparatus employed in lipoprotein/exosome isolation experiments.

Figure 2.

Chromatograms of LDL, plasma-based exosome standard, and LDL+ exosome standard isolation. a) UV absorbance at 216 nm; b) Fluorescence excitation at 500 nm and emission at 520 nm. Step gradient of 25% glycerol with 1M (NH₄)₂SO₄ (3 min) (fraction 1) and 50% glycerol (5 min) (fraction 2) were performed. Separations were performed with a mobile phase flow rate of 0.5 mL min⁻¹. Injection volume = 40 μL. LDL concentration = 0.25 mg mL⁻¹ and exosome standard density = 2.9 × 10¹² particles mL⁻¹.

Figure 3.

Bioinformatics analysis. a) Venn diagram showing the overlap of proteins identified in this study versus the Vesiclepedia database. b) Gene ontology analysis of all identified proteins for molecular function and components of the LDL sample in fraction 2.

Figure 4.

TEM images of the raw LDL and exosome solutions, the mixture, and fractions collect following the protein elution (fraction 1) and exosome elution (fraction 2) steps. a-c) LDL standard, d-f) exosome standard, and g-i) LDL and exosome mixture. In each case, the scale bars represent a length of 200 nm.

Figure 5.

Indirect ELISA employing antibodies to the EV marker - CD81 (a) and LDL marker - Apo-B 100 (b) proteins to confirm the presence of LDLs and EVs in the sample standards, and after recovery from the C-CP tip using the HIC mode workflow. The elutions were recovered from protein (P - 25% glycerol, 1M ammonium sulfate) (fraction 1) and EV (E - 50% glycerol) (fraction 2) elution conditions.