## 6 Extracellular-Vesicle Pharmacokinetics  
As explained in the previous section, different studies have shown that MSC-EV are involved in various biological processes. For this reason, since they are efficient vehicles to transport nucleic acids, proteins and other bioactive compounds, MSC-EV are expected to be used in therapeutic treatments, such as tissue regeneration. To develop therapies based on these vesicles, it is important to know their pharmacokinetics when they are applied in vivo. To elucidate the pharmacokinetics of MSC-EV, their tissue distribution must be evaluated. For this purpose, different methods of labeling exogenously administered EV have been used. One of them is the use of lipophilic fluorescent dyes to label EV for in vivo monitoring purposes. For example, it has been observed that EV derived from intravenously administered murine B16F10 melanoma cells accumulated in bone marrow, spleen, liver and lung, due to the lipophilic fluorescent dye PKH67. Furthermore, different studies in animal models observed that MSC-EV labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindolicarbocyanine perchlorate (DiD), and intravenously injected, were distributed throughout spleen, liver and kidney [75]. Moreover, EV derived from different cells and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylimidotricarbocyanine iodide (DiR) allowed us to elucidate the in vivo behavior of such vesicles [76]. Thus, EV were mainly distributed in the spleen, liver, lung and gastrointestinal tract when intravenously injected. Notably, tissue distribution of EV derived from HEK293T human embryonic kidney cells was different, depending on the injection method. They were mainly stored in the liver when intravenously applied. However, vesicles administered by subcutaneous or intraperitoneal injection accumulated in the pancreas, liver and gastrointestinal tract. The biodistribution of EV derived from body fluids, such as milk, has also been studied [77]. These vesicles were labeled with DiR and orally or intravenously applied. They are distributed into various organs such as the lung, kidney, liver, spleen, pancreas, colon, brain and ovaries after four days, when orally administered. However, exosomes intravenously injected accumulated in the liver. Such a result is in line with what has been demonstrated in other studies of EV derived from cultured cells. Another aspect to be considered for the study of pharmacokinetics is the time of elimination from the bloodstream, and accumulation in organs. It was observed by bioluminescence that the half-life of gLuc-LA-labeled B16 BL6 EV was approximately two minutes after intravenous injection. Furthermore, it was found that these EV were mainly distributed in the lung, liver and spleen. Also, it was observed that EV derived from different mouse cell lines have a half-life of two to four minutes, after intravenous injection, being accumulated in the liver [78; 79]. These studies demonstrate that there are several types of EV target tissues although, regardless of the method of exosome labeling, intravenously administered EV rapidly disappear from blood circulation, being mainly stored in the liver. It is also important to identify which cell types recognize and take up EV, incorporating their cargos, for further advancement in the development of EV-based therapies. Different studies indicate that the primary cells that actively take up exogenous EV are macrophages. Thus, clearance of intravenously injected B16 BL6 EV from the bloodstream was slower in mice without macrophages, thus marking the importance of these cells in the pharmacokinetics of exosomes. It has been observed that macrophages are responsible for hepatic and splenic uptake of B16 BL6 EV, while in the lung they were mainly taken up by endothelial cells [78]. To address the problem of rapid clearance of EV when intravenously, subcutaneously or intraperitoneally administered, a combination of vesicles with biomaterials, such as hydrogels, has become the main research focus of exosome-based therapies.