Microvesicle-mediated RNA molecule delivery system using monocytes/macrophages

Abstract

Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, are possible vehicles for navigating RNA molecules to body tissues. It is considered that intravenous injection of such MVs or exosomes from patients would not cause severe not-self and toxic reactions. Previously, we found that macrophages take up liposome-entrapped RNA molecules, some of which remain undegraded in the cells. Here, we demonstrate that transfected RNA molecules in human monocytic leukemia THP-1 cells were shed from THP-1 macrophages as contents in MVs during incubation in serum-free medium, which shedding was shown by biochemical analyses such as quantitative reverse transcription (qRT)-PCR, expression of TSG101 (a membrane-associated exosomal protein), and immunoelectron microscopic study. More chemically modified RNA molecules (miR-143BPs) entrapped by MVs (MV-miR-143BPs) were secreted from THP-1 macrophages after miR-143BP transfection compared with the amount after transfection with nonmodified miR-143 transfection. Furthermore, we show that the THP-1 macrophages, which were transfected with the miR-143BP ex vivo, secreted MV-miR-143BPs in xenografted nude mice after intravenous injection, because miR-143 levels were significantly increased in the serum, tumor, and kidney of the host animals. These data suggest that some of the transfected miR-143BPs were secreted from THP-1 macrophages as MV-RNAs both in vitro and in vivo.

Figure 1

Comparison of miR-143 levels in the intracellular fraction from THP-1 macrophages and in shed microvesicles (MVs) after transfection with chemically modified miR-143BP or nonmodified miR-143. (a) miR-143 levels in the intracellular fraction of THP-1 macrophages. The relative amount of miR-143 is shown. The miR-143 level in the nonspecific control (C) is indicated as 1. (b) miR-143 levels in the MV-rich fraction of THP-1 macrophages. The relative amount of miR-143 is shown. Again, the miR-143 level in the control is indicated as “1”. The procedure for preparation of the MV-rich fraction is described in Materials and Methods section. (c) miR-143 levels in the intracellular fraction from THP-1 cells and their differentiated macrophages after transfection with miR-143BP in the presence of serum in the medium. The relative amount of miR-143 is shown. The miR-143 level in the control is indicated as “1”. The levels of miR-143 were evaluated by performing a TaqMan miRNA assay using real-time PCR. THP, human acute monocytic leukemia cell line.

Figure 2

Biochemical detection of miR-143 in the MV-rich fraction from THP-1 macrophages after transfection with miR-143BP and incubation in serum-free medium. Detection was made by performing the TaqMan miRNA assay using real-time PCR and western blot analysis of TSG101. (a) The relative amount of miR-143 is shown. The miR-143 level in the NC control (C) is indicated as “1”. miR-21 was used as an internal control. (b) The relative amounts of GAPDH and β-actin are shown. (c) Western blot analysis of TSG101. Ten micrograms of protein from THP-1 cells (macrophages) or the MV-rich fraction was loaded into each lane. The ratios of Tsg101/β-actin, which were evaluated by densitometry, are also shown. The procedure for preparation of the MV-rich fraction is described in Materials and Methods section. MV, microvesicle; THP, human acute monocytic leukemia cell line.

Figure 3

Ultrastructural detection of miR-143 in the MV-rich fraction. (a) MVs observed by electron microscopy in the MV-rich fraction. (b,c) Immunoelectron microscopic study. Immunogold-anti-Cy5 was used for the detection of Cy5, with which miR-143BP had been labeled. The MVs in the (b) multivesicular body and (c) extracellular space are shown, and the specific signals (gold particles) indicating miR-143BP/Cy5 are indicated by the arrows. There is no signal in the controls (upper photos). MV, microvesicle.

Figure 4

Tissue distribution of miR-143 in human colon cancer DLD-1 cell/xenograft nude mice after the intravenous injection of THP-1 macrophage-enriched suspension after the ex vivo transfection of the cells with miR-143BP. (a) Relative amounts of miR-143 are shown in the tumors and kidneys. The miR-143 level in the chemically modified NS/BP control (C) is indicated as “1”. The miR-143 level was evaluated by TaqMan miRNA assay using real-time PCR. (b) The time course of the serum level of miR-143 after the last injection of the miR-143BP/THP-1 macrophage-enriched suspension into the xenografted nude mice. The procedure for the in vivo experiment is described in Materials and Methods section. THP, human acute monocytic leukemia cell line.

Figure 5

Schema of microvesicle-mediated RNA molecule delivery system using monocytes/macrophages for clinical application. iPS, induced pluripotent stem cell; MV, microvesicle.