In vivo Non-invasive Imaging of Radio-Labeled Exosome-Mimetics Derived From Red Blood Cells in Mice

Abstract

Exosomes are natural nano-sized membrane vesicles that have garnered recent interest owing to their potential as drug delivery vehicles. Though exosomes are effective drug carriers, their production and <i>in vivo</i> biodistribution are still not completely elucidated. We analyzed the production of exosome mimetics (EMs) from red blood cells (RBCs) and the radio-labeling of the RBC-EMs for <i>in vivo</i> imaging. Engineered EMs from RBCs were produced in large-scale by a one-step extrusion method, and further purified by density-gradient centrifugation. RBC-EMs were labeled with technetium-99m (^99mTc). For non-invasive imaging, ^99mTc (free) or ^99mTc-RBC-EMs were injected in mice, and their biodistribution was analyzed by gamma camera imaging. Animals were sacrificed, and organs were collected for further biodistribution analysis. RBC-EMs have similar characteristics as the RBC exosomes but have a 130-fold higher production yield in terms of particle numbers. Radiochemical purity of ^99mTc-RBC-EMs was almost 100% till 2 h reduced to 97% at 3 h. Radio-labeling did not affect the size and morphology of RBC-EMs. In contrast to free ^99mTc, <i>in vivo</i> imaging of ^99mTc-RBC-EMs in mice showed higher uptake in the liver and spleen, and no uptake in the thyroid. <i>Ex vivo</i> imaging confirmed the <i>in vivo</i> findings. Furthermore, fluorescent imaging confirmed the nuclear imaging findings. Immunofluorescent imaging revealed that the hepatic uptake of RBC-EMs was significantly mediated by kupffer cells (resident hepatic macrophages). Our results demonstrate a simple yet large-scale production method for a novel type of RBC-EMs, which can be effectively labeled with ^99mTc, and feasibly monitored <i>in vivo</i> by nuclear imaging. The RBC-EMs may be used as <i>in vivo</i> drug delivery vehicles.