Subject Number : S-16-NM-0053
Support Type : 機器利用
Proposal Title (English) : Study endocytosis, exocytosis and drug release in exosome
Username (English) : Yan-Jye Shyong,
Affiliation (English) : Institute of Biomedical Engineering, National Taiwan University

1. Summary
Exosomes are attractive as potential carriers for drug delivery because of their natural function of transferring biomolecules among cells without eliciting immune responses. However, an obstacle to the application of exosomes for drug delivery is the difficulty in collecting sufficient numbers of these vesicles. We found that treatment with calcium phosphate (CaP) particles increased the number of exosomes secreted from macrophage-like RAW264.7 cells and monocyte-like THP-1 cells. CaP particles were easily internalized into cells and dissolved in acidic late-endosomes or lysosomes, resulting in the rupture of their membranes followed by the release of Ca2+ into cytosol. However, the Ca2+ concentrations in exosomes secreted from CaP particle-treated cells were similar to that in exosomes from untreated control cells, implying that exosomes secreted from cells treated with CaP particles are not contaminated by the Ca2+ released from CaP particles. This study highlights the potential for the efficient production of exosomes using CaP particles.

2. Experimental
Mainly used facility: Cell culture facility, laser confocal fluorescence microscope and table top SEM.
CaP is made by co-precipitation method and viewed by SEM. Cell uptake of CaP is viewed by confocal. Cell cytotoxicity is done by WST-8. Exosome is isolated by Total Exosome Isolation reagent and analysied by CD9 and LAMP1 antibody. Amount of exosome is measured by EXOCET assay. Finally, the calcium concentration in exosome is measured by ICP.
Facility staffs assisted training of SEM, cell culture and conforcal microscope. Also assist me on the experiment design.

3. Results and Discussion
CaP particles increased the number of exosomes secreted from phagocytes such as macrophages. CaP particles were localized in late-endosomes or lysosomes, but CaP particles dissolved in these acidic vesicles, leading to the release of Ca2+ into cytosol by the rupture of their membranes. Most exosomes were secreted after the release of dissolved Ca2+ into cytosol and secreted exosomes did not contain Ca2+, which suggests that exosomes were formed before the release of dissolved Ca2+ into cytosol. Recently, exosomes have been studied for the application to drug carrier, but it is difficult in collecting sufficient numbers of exosomes. This study demonstrated that CaP particles have a potential to improve the production efficiency of exosomes that are not contaminated by Ca2+.

4. Others
This work was supported by the international cooperative graduate program between the National Institute for Materials Science (NIMS), Japan, and the National Taiwan University (NTU), Taiwan. Mrs. Akio Iwanade and Akira Ishitoya, Materials Analysis Station, National Institute for Materials Science, are acknowledged for support of ICP-OES experiment

5. Publication/Presentation
(1) Y.J. Shyong, Calcium phosphate particles stimulate exosome secretion from phagocytes, 6th Annual World Congress of Nano Science and Technology, Singapore.

(2) Y.J. Shyong, Calcium phosphate particles stimulate exosome secretion from phagocytes,10th Anniversary International Symposium on Nanomedicine, Japan.

6. Patent
N/A