(662a) Real-Time Characterization of Exosomes Secreted From Single Cells by Tethered Lipoplex Nanoparticles and TIRF Microscopy

Exosomes are small membrane vesicles with diameter of 50–100 nm secreted by a wide range of cells. Exosomes arise from the endocytic cellular pathway and are released when multivesicular bodies fuse with the plasma membrane. Exosomes can mediate communication between cells, facilitating processes such as antigen presentation. In 2007, exosomes were found to be natural carriers of over 1300 messenger RNAs (mRNA) and 121 non-coding microRNAs (miRNA), a major breakthrough in understanding exosome biology. Recently circulating exosomes have been demonstrated to be a potiental biosignature for cancer early diagnosis.

Although significant progress has been made, the biological function of exosomes is only just beginning to be understood. Currently, exosomes are isolated by ultracentrifugation and the encapsulated mRNA and miRNA expressions are characterized by qRT-PCR. These methods are time consuming and labor intensive. More importantly, information collected from these conventional methods is an average from a large number of exosomes. We have developed a tethered lipoplex nanoparticles based real-time imaging platform to visualize and characterize individual exosomes right after they are released by the cells. This platform was demonstrated with A549 non-small cell lung cancer cells and a microRNA, miR-21, was selected as the biomarker. Briefly, A549 cells were seeded on a glass substrate. Molecular beacons that detect miR-21 were encapsulated in cationic lipoplex nanoparticles tethered around the A549 cells. When exosomes were released from A549 cells, the electrostatic force between negatively charged exosomes and positively charged lipoplex nanoparticles facilitated the fusion between the naturally occurred nanoparticles and man-made nanoparticles. The molecular beacon encapsulated in the lipoplex nanoparticles then detected miR-21 in the exosomes. Total internal reflection fluorescence (TIRF) microscopy was used to capture the fluorescence signal emitted from the molecular beacon and detect the copy numbers of miR-21 in each exosome.

This is the first platform that is capable of visualizing and characterizing exosomes in real time. This technique may also be used to study cell-cell communication and cell response to the stimuli, such as anti-cancer drugs or the change of microenvironment.