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Lipid Nanoprobe Method Enables Rapid Isolation of Extracellular Vesicles

By BiotechDaily International staff writers
Posted on 25 Apr 2017
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Image: Lipid nanoprobes (blue, green, and yellow colored) spontaneously insert themselves into lipid bilayer of three extracellular vesicles. The cargo content of extracellular vesicles includes proteins, DNA, and RNA. The lipid nanoprobe-labeled extracellular vesicles are captured onto the surface of a magnetic bead (black, bottom) through interaction with conjugated avidin molecules (red) (Photo courtesy of Xin Zou / Pennsylvania State University).
Image: Lipid nanoprobes (blue, green, and yellow colored) spontaneously insert themselves into lipid bilayer of three extracellular vesicles. The cargo content of extracellular vesicles includes proteins, DNA, and RNA. The lipid nanoprobe-labeled extracellular vesicles are captured onto the surface of a magnetic bead (black, bottom) through interaction with conjugated avidin molecules (red) (Photo courtesy of Xin Zou / Pennsylvania State University).
A novel method reduces the amount time required to isolate extracellular vesicles (EVs) from culture media or blood plasma from hours to 15 minutes and does not require bulky or expensive equipment.

EVs, which include exosomes, microvesicles, and apoptotic bodies, are cell-derived lipid-bilayer-enclosed structures, with sizes ranging from 30 to 5,000 nanometers. In the past decade, EVs have emerged as important mediators of cell communication because they serve as vehicles for the intercellular transmission of biological signals (proteins or nucleic acids) capable of altering cell function and physiology. In particular, exosomes (EVs with diameters of 30 to 150 nanometers) containing cell and cell-state specific proteins and nucleic acids are secreted by many cell types and have been identified in diverse body fluids. Growing evidence indicates that nanoscale EVs (nEVs) can regulate tumor immune responses, initiate formation of the pre-metastatic niche, determine organotropic metastasis, and contribute to chemotherapeutic resistance. nEVs are thus potential targets for therapeutic intervention in cancer.

While it is highly desirable to isolate nEVs rapidly for downstream molecular analyses, approaches reported for the isolation of nEVs - such as ultracentrifugation, immunoisolation, polymer-based precipitation, and filtration - involve lengthy protocols and can lead to impurities and nEV damage.

Investigators at Pennsylvania State University have developed a new technique to expedite isolation of nEVs. This lipid nanoprobe (LNP) system involves the labelling of the lipid bilayer of nEVs with biotin-tagged 1,2-distearoyl-sn-glycero-3-phosphethanolamine-poly(ethylene glycol) (DSPE–PEG). The labelled nEVs are then collected by NeutrAvidin (NA)-coated magnetic sub-micrometer particles (MMPs), for subsequent extraction and analyses of nEV cargo.

Compared with differential centrifugation (the most prevalent method for nEV isolation), the LNP shortens the isolation procedure from hours to 15 minutes and does not require bulky or expensive equipment. It is also highly flexible and can be adopted for various downstream analyses of DNA, RNA, and proteins.

The investigators reported in the April 10, 2017, online edition of the journal Nature Biomedical Engineering that they had applied the LNP method to obtain nEV DNA from 19 stage-IV non-small-cell lung-cancer (NSCLC) patients. Analyses of the contents of the nEVs allowed the detection of mutations in KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) codons 12 and 13 and EGFR (epidermal growth factor receptor) exons 19 and 21.

“We invented a system of two micro/nano materials,” said senior author Dr. Si-Yang Zheng, associate professor of biomedical engineering and electrical engineering at Pennsylvania State University. “One is a labeling probe with two lipid tails that spontaneously insert into the lipid surface of the extracellular vesicle. At the other end of the probe we have a biotin molecule that will be recognized by an avidin molecule we have attached to a magnetic bead. Most cells generate and secrete extracellular vesicles, but they are difficult for us to study. They are sub-micrometer particles, so we really need an electron microscope to see them. There are many technical challenges in the isolation of nanoscale EVs that we are trying to overcome for point-of-care cancer diagnostics.”


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