Direct isolation and characterization of circulating exosomes from biological samples using magnetic nanowires
- 444 Downloads
Tumor-derived exosomes are gaining attention as important factors that facilitate communication between neighboring cells and manipulate cellular processes associated with cancer development or progression. The conventional techniques for the isolation and detection of exosomes face several limitations, restricting their clinical applications. Hence, a highly efficient technique for the isolation and identification of exosomes from biological samples may provide critical information about exosomes as biomarkers and improve our understanding of their unique role in cancer research. Here, we describe the use of antibody cocktail-conjugated magnetic nanowires to isolate exosomes from plasma of breast and lung cancer patients.
The isolated exosomes were characterized based on size and concentration using nanoparticle tracking analysis. Levels of exosomal proteins were measured by bicinchoninic acid assay and enzyme-linked immunosorbent assay. Morphology was visualized by transmission electron microscopy. Immunoblotting (Western blotting) was used to detect the presence of exosomal markers.
The use of antibody cocktail-conjugated magnetic nanowires resulted in approximately threefold greater yield when compared to the conventional methods. The elongated feature of nanowires significantly improved the efficiency of exosome isolation, suggesting its potential to be translated in diverse clinical applications, including cancer diagnosis and treatment.
KeywordsPlasma Exosome Magnetic nanowire Lung cancer Breast cancer
the magnetic nanowires conjugated with a large amount of magnetic nanoparticles and antibody cocktails
circulating tumor cells
anodized aluminum oxide
scanning electron microscopy
transmission electron microscopy
concentrated culture medium
nanoparticle tracking analysis
quantitative reverse transcription polymerase chain reaction
Results and discussion
Preparation and characterization of antibody cocktail-conjugated magnetic nanowires (Abs_MNWs)
We have recently demonstrated MNWs as a highly efficient platform for the capture and enrichment of CTCs and cfDNA from biological samples (blood or urine) of cancer patients . The nanowire-based approach can greatly improve the recovery yield and purity by specifically enhancing the interaction with tumor-specific biomarkers in biologically complex fluids. The enhanced interaction is most likely attributed to the following inherent topographic features of nanowire: (i) a large surface area that allows direct incorporation of, or modification with, available functional groups; (ii) long and thin morphology that assists in detection and capture of tumor-associated markers, with minimum steric hindrance between the nanostructure and other components present in the blood or urine; and (iii) the elongated structure that is capable of encapsulating large amounts of iron oxide nanoparticles (~ 10 nm) in its interior during electrochemical deposition, which has significant impact on the magnetic response of the resulting nanowires.
Circulating tumor-derived exosomes are known to play a key role in the process of carcinogenesis. As exosomes are released at a high level during cancer progression, exosome amount detected from cancer patients is much higher than that from healthy individuals. Thus, exosomes can serve as a valuable biomarker with significant clinical relevance in both biological and clinical research. Despite tremendous progress in the exosome extraction techniques, there is a need for standard optimized protocols. We applied the nanowire-based strategy for the isolation and purification of exosomes from the plasma of cancer patients. The SS-biotin- and MNP-doped Ppy nanowires were electrochemically deposited within the well-ordered nanoporous AAO template using a mixture of pyrrole monomers, MNPs, and SS-biotin. After removing the AAO template completely, the resulting MNWs were further labeled with a cocktail of antibodies that have been considered as a versatile and efficient platform for exosome capture as well as DTT-mediated release of the captured exosome with ease, robustness, and efficiency (Fig. 1a; Abs_MNWs). Thin elongated Abs_MNWs are capable of providing sufficient binding sites to covalently link antibodies specific to exosomes (anti-CD9, anti-CD63, and anti-CD81) via biotin-streptavidin interactions. As members of the tetraspanin family of proteins, CD9, CD63, and CD81 are overexpressed in exosomes, predominantly located on the surface, thereby serving as a potential exosomal marker. Abs_MNWs may offer several advantages in the extraction and identification of exosomes with phenotypic variation while reducing the inevitable loss of circulating exosomes during the capture process. MNWs displayed an average length of 18 μm and diameter of 200 nm, as observed by SEM and TEM (Fig. 1b; left and middle). The assemblies of randomly distributed, highly packed MNPs embedded in Abs_MNWs were revealed by TEM image (Fig. 1b; right). As a result of the high density of MNPs, MNWs can possess a high saturation magnetization (Ms = 57 emu/g), while no magnetic response was observed with bare nanowires (Fig. 1c).
Analysis of exosomes isolated from cell lines by magnetic beads and magnetic nanowires
Evaluation of exosomes collected from the plasma of healthy donors and patients with breast and lung cancer using Abs_MNWs
Summary of the characteristics of the Abs_MNWs method for exosome isolation
(1) Exosome capture by Abs_MNWs, 30 min
(2) Place the tube (1) in a magnetic separation rack and wash with PBS, 5 min
(3) Elute exosomes from Abs_MNWs by incubating in DTT solution, 25 min
Cost (per 1 mL sample)
Minimum volume of plasma
Thus, nanowire-based method allows isolation of homogeneous population of exosomes with higher yield and purity and displays potential applications for the analysis of protein, lipid, mRNA, and miRNA from highly purified exosomes. This may be useful in studying the biological functions and role of exosomes in cancer development.
We demonstrate a novel approach for efficient isolation and detection of exosomes using Abs-MNWs coupled to different types of exosome-specific antibodies and high density of MNPs. The elongated morphology of nanowires affords more flexibility and versatility for exosome isolation and identification by facilitating multiple interactions through recognition receptors on exosomes, thereby resulting in enhanced exosome recovery even from small volumes of blood plasma of cancer patients. Overall, the simplicity of preparation and excellent performance of the nanowire-based strategy offer high sensitivity and specificity in exosome isolation and detection, which can be widely applied to a variety of cancer types for cancer screening and diagnosis.
Chemicals and reagents
Pyrrole, poly(sodium 4-styrenesulfonate) (PSS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), iron oxide (II, III), MNP solution (average diameter, 10 nm), streptavidin, and sodium hydroxide (NaOH) were obtained from Sigma Aldrich (St. Louis, MO, USA). An anodized aluminum oxide (AAO) membrane filter (pore diameter, 200 nm) was purchased from Whatman (Pittsburgh, PA, USA). NHS-SS-biotin was supplied by CovaChem (Loves Park, Illinois, USA). Biotinylated anti-CD63 and anti-CD81 were obtained from AnCell (Oak Park, Minnesota, USA). Biotinylated anti-CD9 was procured from Abcam (Cambridge, UK). Anti-CD9, anti-CD63, anti-CD81 was purchased from Cell Signaling Technology (Denver, MA, USA). Invitrogen™ Exosome-Human CD9 Isolation Reagent from cell culture (Dyna Beads_CD9) and Invitrogen™ Exosome-Human CD81 Isolation Reagent from cell culture (Dyna Beads_CD81) were obtained from ThermoFisher Scientific Inc. (Waltham, MA, USA).
Fabrication and characterization of anti-CD9-conjugated, anti-CD81-conjugated, and antibody cocktail-conjugated magnetic nanowires (CD9_MNWs, CD81_MNWs, and Abs_MNWs)
We prepared CD9_MNWs, CD81_MNWs, and Abs_MNWs as previously described . Briefly, MNPs (average diameter, 10 nm) were incubated inside the pore of Au-coated AAO membrane filter (pore diameter, 200 nm), followed by gentle aspiration. Electrochemical experiments were conducted using a potentiostat/galvanostat (BioLogic SP-50), with a Pt wire, Ag/AgCl reference, and Au-coated AAO membrane used as a counter, reference, and working electrode, respectively. Polypyrrole (Ppy) was electrochemically polymerized to the pores of AAO membrane in a solution containing 0.1 M pyrrole, 0.01 M PSS, and 1 mM NHS-SS-biotin by applying chronoamperometry (CA) at 1.5 V for 7 min. The resulting AAO membranes were washed several times with distilled water and incubated in 2 M NaOH for 2 h to remove AAO template. In the subsequent steps, 6 mM NHS and 30 mM EDC were added to the resulting MNWs and incubated for an additional 45 min. MNWs were immersed in streptavidin (10 µg/mL) for 45 min at room temperature, followed by washing with water. After streptavidin labeling, the biotinylated antibody cocktail (i.e., biotinylated anti-CD9, biotinylated anti-CD63, and biotinylated anti-CD81 in Dulbecco’s phosphate-buffered saline) was conjugated to streptavidin-labeled MNWs at 4 °C overnight to obtain the final product (i.e., Abs_MNW) with a final antibody concentration of 0.4 µg/mL. For the preparation of anti-CD81 or anti-CD9 conjugated magnetic nanowires, biotinylated anti-CD81 or biotinylated anti-CD9 was linked to streptavidin-labeled MNWs at 4 °C overnight to produce the final product (i.e., CD81_MNWs or CD9_MNWs) with a final antibody concentration of 0.4 µg/mL. The mean number of antibodies bound per MNW was determined using a previously described assay . Briefly, we detected and quantified the amount of antibodies conjugated onto 1.26 × 106 MNWs/mL by incubating them with horseradish peroxidase-labeled (HRP) anti-mouse IgG for 1 h, where a 3% solution of bovine serum albumin (BSA) was employed to prevent nonspecific binding. MNWs were washed carefully several times to remove any unbound IgG. HRP attached to the MNWs was further treated with 3,3′,5,5′-tetramethyl benzidine, producing a colored product; the relative amount of antibody was determined in comparison with an HRP anti-mouse IgG standard curve. The results were read using a spectrophotometer at 650 nm. The morphology of Abs_MNW was observed by scanning electron microscopy (SEM; JSM-6701F, JEOL) with an accelerating voltage of 15 kV and transmission electron microscopy (TEM; G2F30, Tecnai) with an accelerating voltage of 300 kV. Magnetic measurements were carried out using a SQUID-VSM magnetometer (MPMS-VSM, Quantum Design, San Diego, CA, USA) with the applied magnetic field in the range from 70 to − 70 kOe.
Cell culture and preparation of concentrated culture medium (CCM)
Four different types of cancer cell lines (MDA-MB-231 and MCF7 breast cancer cells, HCT116 colon cancer cells, and HeLa cervical cancer cells) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in a 5% CO2 atmosphere. The cells (~ 2 × 109 cells) were pelleted and washed thrice with RPMI-1640 media, followed by replacement of media with serum-free RPMI media. The cells were cultured for additional 2 days in serum-free RPMI media before exosome harvesting. Intact cells and cell debris were removed by centrifugation at 300×g for 10 min and 2000×g for 20 min, respectively. CCM was collected and filtered through sterile 0.22-µm (pore-size) syringe filter (Merck Millipore, USA) [2, 4, 14, 25].
Exosome isolation by Dyna Beads_CD9, Dyna Beads_CD81, CD9_MNWs, CD81_MNWs, and Abs_MNWs
For isolation of circulating exosomes, Dyna Beads_CD9 (5.0 × 105 Beads/µL), Dyna Beads_CD81 (5.0 × 105 Beads/µL), CD9_MNWs (1.0 × 103 MNWs/µL), CD81_MNWs (1.0 × 103 MNWs/µL), and Abs_MNWs (1.0 × 103 MNWs/µL) were incubated in 250 µL–3 mL CCM or plasma of healthy donors and cancer patients for 30 min at room temperature with gentle shaking to promote attachment of exosomes. Next, a magnetic field created by the MagneSphere® Technology Magnetic Separation Stands (Promega, USA) was applied on the sample tubes (1.5 mL microcentrifuge tubes) to efficiently remove the supernatant and collect the captured exosomes. Dithiothreitol (DTT) solution (50 mM) was added to the resulting solution to release the captured exosomes from the nanowires by breaking disulfide bonds. We evaluated the concentration and size of exosomes isolated by MNWs using the nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Instruments, Malvern, UK) and Malvern Zetasizer Nano-Z (Malvern Instruments, Malvern, UK). In addition, total protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, 1 µL of isolated exosome was diluted in 19 µL of M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA) and 200 µL of BCA reagent A and B mixture (A:B = 50:1) was added and incubated for 30 min at 37 °C. The optical density (OD) of the sample was measured by a UV/VIS spectrophotometer at a wavelength of 562 nm. The protein concentration was calculated from standard BCA curve (r2 = 99.8%). All measurements were carried out under constant experimental conditions to obtain comparable results. For exosome sandwich ELISA assay, 100 µL of anti-CD9 antibody (1 µg/100 µL) was coated onto 96 well plate (Thermo Fischer Scientific) and incubated at 4 °C overnight. Then, the plate was blocked with 1% BSA in PBS buffer at 37 °C for 1 h. After washing with 0.1% BSA-PBS buffer three times, the plate was incubated with an exosome solution in PBS buffer (100 μL) at 37 °C for 1 h. Upon removing the solution, the plate was washed twice with 0.1% BSA-PBS buffer and added to biotin-conjugated detection antibodies (anti-CD81; LifeSpan Biosciences, Inc., Seattle, WA, USA) in PBS buffer (100 μL; 500 ng/mL), followed by incubating at room temperature for 1 h. After washing three times with 0.1% BSA-PBS buffer, the plate was incubated again with a solution of HRP conjugated streptavidin in PBS buffer (100 μL; 1:1000) at room temperature for 30 min and then washed three times with 0.1% BSA-PBS buffer. TMB Ready Solution (Thermo Fisher Scientific) was then added to the plate and incubated at room temperature for 15 min, followed by the addition of 50 μL of stop solution to each well. The absorbance was read using a UV/VIS spectrophotometer at a wavelength of 450 nm.
Exosome isolation by commercial extraction kits
Exosomes were isolated and purified using ExoQuick (EXOQ5TM-1, System Biosciences, Palo Alto, CA, USA), Invitrogen Total Exosome Isolation Kit (4484451, Thermo Fisher Scientific, Massachusetts, Waltham, USA), and Exosome-Human CD81 Flow Detection Reagent (10622D, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, the reagents were added to CCM or plasma of healthy donors and cancer patients to isolate exosomes and the mixture was vortexed and centrifuged at 4 °C as described in the manufacturers’ protocols. The pellet containing exosomes was resuspended in DPBS or ultrapure water. Subsequently, the exosome pellet was diluted in M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA) and BCA reagent A and B (A:B = 50:1) was added and incubated for 30 min at 37 °C. The protein concentration of the pellet was determined using the BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. To improve reproducibility, all assays were carried out under same experimental conditions.
Blood specimen collection and preparation
Whole blood was collected in Vacutainer tubes containing the anti-coagulant ethylenediaminetetraacetic acid (EDTA), following procedures approved by the National Cancer Center Institutional Review Board. The collected blood was centrifuged at 3000×g for 10 min for separation of plasma, which was stored at − 80 °C until analysis.
Transmission electron microscopy (TEM) analysis of exosomes
Freshly isolated exosomes from cells were resuspended in cold DPBS. Exosome samples were prepared for TEM analysis using exosome-TEM-easy kit (101Bio, Palo Alto, CA, USA) according to the manufacturer’s instruction. Briefly, re-suspended exosomes were mounted on Formvar-carbon coated EM mesh 400 grids and incubated for 10 min. The resulting grids were rinsed twice with wash buffer and deposited on the EM solution for 10 min. After washing and dehydration steps, exosomes were subjected to TEM with an accelerating voltage of 300 kV.
Fluorescence analysis of exosomes
The captured and released exosomes on/from the Abs_MNWs were labeled with a fluorescent Vybrant™ DiO dye solution (5 µL/mL, Molecular Probes, Life Technologies) by incubating for 8 min at 37 °C to allow staining of the exosomal membrane. The exosomes were rinsed with phosphate-buffered saline (PBS) and the DiO-labeled exosomes were analyzed by a Zeiss fluorescence microscope.
Exosomes isolated by Abs_MNWs were lysed in M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA). Protein samples (20 µg) were separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 µm, Millipore). The membranes were blocked with 3% skim milk for 1 h at room temperature and probed with primary mouse anti-TSG101 (1:1000), rabbit anti-HSP70 (1:1000), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (1:1000), rabbit anti-CD9 (1:1000), rabbit anti-CD63 (1:1000), rabbit anti-CD81 (1:1000), and rabbit monoclonal anti-GAPDH (1:1000) for overnight. Following incubation, the membranes were incubated with an appropriate secondary antibody (goat anti-mouse IgG [1:3000] or goat anti-rabbit IgG [1:3000]) for 1 h. Blots were washed three times with TBST buffer after each incubation step and visualized using a SuperSignal® West Pico Chemiluminescent Substrate reagent (34077, Thermo Scientific).
RNA extraction and miRNA analysis
Total exosomal RNA was extracted with TRIzol (Invitrogen, Paisley, UK) and homogenized through a pipette following the manufacturer’s protocol. Further, RNA samples were treated with chloroform (Merck, Darmstadt, Germany) and centrifuged for 15 min at 12,000×g at 4 °C to separate the mixture into aqueous and organic phases, and isopropanol was used to precipitate the supernatant. Then, miR21-specific complementary DNA (cDNA) was synthesized from 10 ng of RNA eluate with random hexamers using TaqMan MicroRNA reverse transcription Kit (Applied Biosystems, Foster city, CA, USA). The quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using an LC480 real-time PCR system (Roche, Basel, Switzerland) for 5 min at 25 °C, 20 min at 46 °C, and 1 min at 95 °C, followed by storage at 4 °C. We used pre-designed primers and probe (TaqMan™ Advanced miRNA Assay, Thermo Fisher Scientific) for miR-21 (has-miR-21-3p, assay ID 477973_mir), prepared according to the manufacturer’s instructions.
JL, MC, YL, and HL performed the experimental work. MC and YC contributed to the analysis and representation of data. YC wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Consent for publication
All authors agree to be published.
Ethics approval and consent to participate
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2017R1A2B4007800) and a Grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: HI17C0828).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 14.Baranyai T, Herczeg K, Onódi Z, Voszka I, Módos K, Marton N, Nagy G, Mäger I, Wood M, Andaloussi S, Pálinkás Z, Kumar V, Nagy P, Giricz Z. Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS ONE. 2015;10:e0145686.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.