Exosomes are Secreted at Distinct Densities in Human Cancer Cells and are Promising Vectors for Hydrophobic Drug Delivery

Abstract

Exosomes are cell-derived vesicles less than 200 nm in size containing gene encoding and gene silencing RNA and cytosolic proteins with roles in intercellular communication. Interest in the use of exosomes as targeted drug delivery vehicles has grown since it was shown that they can bind to specific cells and deliver intact genetic material to the cytosol of target cells. We present the detailed isolation protocol which allowed us to isolate secreted vesicles, including exosomes, from human prostate cancer (PC3) and melanoma (M21) cell lines. Transmission electron microscopy (TEM) showed that the isolated vesicles displayed a cup-shaped morphology as well as prolate shapes previously unreported in the literature. Nanoparticle Tracking Analysis (NTA) and western blot assays demonstrated that these vesicles were largely within the 30 - 200 nm size range expected for exosomes and were enriched for CD63, an exosome marker protein. NTA data further revealed discrete vesicle subpopulations with narrowly defined size ranges and consistent ratios of 30 - 200 nm vesicles to vesicles of all sizes secreted from both cell lines. These findings suggest a cellular control mechanism governing the fraction of secreted vesicles within the 30 - 200 nm range. Our work provides novel insights on exosome biogenesis and highlights the potential advantages of exosomes as hydrophobic drug carriers.