Introduction: The placenta produces three types of extracellular vesicles (EVs): macro-, large- and small-EVs. The in vivo targeting of placental EVs depends on the EV type and exposure time. Since current models predominately involved one-off EV administration, we aimed to elucidate the biodistribution of placental EVs closer to a physiological context where EVs were administered repeatedly to mimic continuous exposure in vivo.
Methods: First-trimester placental tissues were cultured overnight with the fluorescent stain CMTPX (1µg/ml). CMTPX-labelled EVs were then isolated from culture medium by sequential centrifugation at 2000g for macro-EVs, 20000g for large-EVs and 200000g for small-EVs. EVs were resuspended in PBS at 1µg/µl based on their protein content and each EV population was injected into four CD1 female mice at 100µg per dose via a tail vein once daily for five consecutive days. Tissues were collected 1-hour and 1-week post last injection. Cryosections were examined for CMTPX-labelled EVs by scanning fluorescent microscopy. The presence of human-specific DNA was assessed by PCR.
Results: Macro-EVs were localised mainly to the lungs, with less in the kidneys and livers, 1-hour post last injection. However, 1-week later macro-EVs were redistributed to the spleens, along with the hepatic and renal lymph nodes. Large-EVs were found predominantly in the kidneys, livers and spleens 1-hour post final injection, but were more prominent in the lungs and hepatic lymph nodes after 1-week. Small-EVs were localised in the kidneys and hepatic lymph nodes 1-hour post last injection, and their presence increased in the kidneys and renal lymph nodes 1-week later. Finally, human-specific DNA was detected in livers from one of the two mice injected with either large- or small-EVs, and in the lungs from one of the two macro-EV injected mice, collecting at 1-hour but not 1-week after the final injection.
Summary: This study demonstrated that it is safe to repeatedly inject mice with human placental EVs with doses of 100µg total EV proteins. Different sized placental EVs had distinct biodistributions following repeated injections into mice. The presence of human-specific DNA in murine tissues suggested that EVs may be able to deliver placental genes to recipient cells.