Preservation of exosomes at room temperature using lyophilization.

The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by developing a method to preserve exosomes. Although exosomes are generally stored at -80 °C, this temperature is not suitable for their handling or transportation and, therefore, other storage methods are desirable. Lyophilization is a promising storage method that can be used to preserve various substances at room temperature. In this study, we sought to develop a room temperature preservation method for exosomes using lyophilization and compared the properties of the lyophilized exosomes with ones stored at -80 °C. Lyophilization without cryoprotectant resulted in the aggregation of B16BL6 melanoma-derived exosomes, while the addition of trehalose, a cryoprotectant, prevented aggregation during lyophilization. PAGE analysis revealed that the proteins and RNA of exosomes were protected following lyophilization in the presence of trehalose. Lyophilization had little effect on the pharmacokinetics of Gaussia luciferase (gLuc)-labeled exosomes after an intravenous injection into mice. Moreover, it was found that lyophilized exosomes retained the activity of loaded gLuc and immunostimulatory CpG DNA for approximately 4 weeks even when stored at 25 °C. In conclusion, lyophilization with trehalose is an effective method for the storage of exosomes for various applications.

Role of Extracellular Vesicle Surface Proteins in the Pharmacokinetics of Extracellular Vesicles.

Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin α6ß1, an integrin known to be involved in lung targeting, was degraded after ProK treatment. The ProK treatment significantly reduced the lung distribution of EVs after intravenous injection. These results indicate that the surface proteins of EVs such as integrin α6ß1 play some roles in pharmacokinetics in terms of reducing Vd and their distribution to the lung.

Accelerated growth of B16BL6 tumor in mice through efficient uptake of their own exosomes by B16BL6 cells.

Exosomes are extracellular vesicles released by various cell types and play roles in cell-cell communication. Several studies indicate that cancer cell-derived exosomes play important pathophysiological roles in tumor progression. Biodistribution of cancer cell-derived exosomes in tumor tissue is an important factor for determining their role in tumor proliferation; however, limited studies have assessed the biodistribution of exosomes in tumor tissues. In the present study, we examined the effect of cancer-cell derived exosomes on tumor growth by analyzing their biodistribution. Murine melanoma B16BL6-derived exosomes increased the proliferation and inhibited the apoptosis of B16BL6 cells, which was associated with an increase and decrease in the levels of proliferation- and apoptosis-related proteins, respectively. GW4869-induced inhibition of exosome secretion decreased the proliferation of B16BL6 cells, and treatment of GW4869-treated cells with B16BL6-derived exosomes restored their proliferation. Next, we treated B16BL6 tumors in mice with B16BL6-derived exosomes and examined the biodistribution and cellular uptake of these exosomes. After the intratumoral injection of radiolabeled B16BL6-derived exosomes, most radioactivity was detected within the tumor tissues of mice. Fractionation of cells present in the tumor tissue showed that fluorescently labeled exosomes were mainly taken up by B16BL6 cells. Moreover, intratumoral injection of B16BL6-derived exosomes promoted tumor growth, whereas intratumoral injection of GW4869 suppressed tumor growth. These results indicate that B16BL6 cells secrete and take up their own exosomes to induce their proliferation and inhibit their apoptosis, which promotes tumor progression.

Role of Phosphatidylserine-Derived Negative Surface Charges in the Recognition and Uptake of Intravenously Injected B16BL6-Derived Exosomes by Macrophages.

Exosomes are cell-derived extracellular vesicles that function as intercellular delivery carriers. Our previous study demonstrated that macrophages in the liver contributed to the rapid clearance of intravenously administered B16BL6-derived exosomes from the systemic circulation in mice. Phosphatidylserine (PS) may be responsible for this clearance because it is exposed on the surface of exosomes and is recognized by macrophages. In this study, the role of PS exposed on the membranes of exosomes in the uptake of B16BL6-derived exosomes by macrophages was investigated. Negatively charged PS- or phosphatidylglycerol-loaded liposomes suppressed the cellular uptake of PKH67-labeled exosomes by macrophages, whereas phosphatidylcholine-containing liposome did not affect uptake. Subsequently, for the in vivo analysis, exosomes were labeled with Gaussia luciferase, a reporter protein, or (3-125I-iodobenzoyl)norbiotinamide using exosome-tropic fusion proteins comprising the exosome-tropic protein lactadherin. The blood clearance of Gaussia luciferase-labeled exosomes after intravenous injection into mice was significantly delayed by the preinjection of PS- or phosphatidylglycerol-containing liposomes. Moreover, the accumulation of (3-125I-iodobenzoyl)norbiotinamide-labeled exosomes in the liver was decreased by the preinjection of PS-containing liposomes. These results indicate that the negative charge of PS in exosomal membranes is involved in the recognition and clearance of intravenously injected exosomes by macrophages.

Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

Exosomes are small membrane vesicles secreted from cells and are expected to be used as drug delivery systems. Important characteristics of exosomes, such as yield, physicochemical properties, and pharmacokinetics, may be different among different cell types. However, there is limited information about the effect of cell type on these characteristics. In the present study, we evaluated these characteristics of exosomes derived from five different types of mouse cell lines: B16BL6 murine melanoma cells, C2C12 murine myoblast cells, NIH3T3 murine fibroblasts cells, MAEC murine aortic endothelial cells, and RAW264.7 murine macrophage-like cells. Exosomes were collected using a differential ultracentrifugation method. The exosomes collected from all the cell types were negatively charged globular vesicles with a diameter of approximately 100nm. C2C12 and RAW264.7 cells produced more exosomes than the other types of cells. The exosomes were labeled with a fusion protein of Gaussia luciferase and lactadherin to evaluate their pharmacokinetics. After intravenous injection into mice, all the exosomes rapidly disappeared from the systemic circulation and mainly distributed to the liver. In conclusion, the exosome yield was significantly different among the cell types, and all the exosomes evaluated in this study showed comparable physicochemical and pharmacokinetic properties.

Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice.

Previous studies using B16BL6-derived exosomes labelled with gLuc-lactadherin (gLuc-LA), a fusion protein of Gaussia luciferase (a reporter protein) and lactadherin (an exosome-tropic protein), showed that the exosomes quickly disappeared from the systemic circulation after intravenous injection in mice. In the present study, the mechanism of rapid clearance of intravenously injected B16BL6 exosomes was investigated. gLuc-LA-labelled exosomes were obtained from supernatant of B16BL6 cells after transfection with a plasmid DNA encoding gLuc-LA. Labelling was stable when the exosomes were incubated in serum. By using B16BL6 exosomes labelled with PKH26, a lipophilic fluorescent dye, it was demonstrated that PKH26-labelled B16BL6 exosomes were taken up by macrophages in the liver and spleen but not in the lung, while PKH26-labelled exosomes were taken up by the endothelial cells in the lung. Subsequently, gLuc-LA-labelled B16BL6 exosomes were injected into macrophage-depleted mice prepared by injection with clodronate-containing liposomes. The clearance of the intravenously injected B16BL6 exosomes from the blood circulation was much slower in macrophage-depleted mice than that in untreated mice. These results indicate that macrophages play important roles in the clearance of intravenously injected B16BL6 exosomes from the systemic circulation.