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Published on: February 5, 2019
Microvesicle-mediated RNA molecule delivery system using monocytes/macrophages
Yukihiro Akao1, Akio Iio, Tomohiro Itoh
1United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan. yakao@gifu-u.ac.jp
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.
Insights
Chemically modified RNA molecules encapsulated in microvesicles (MVs) were secreted by macrophages. These microvesicle-encapsulated RNAs were detected in host animals after intravenous injection, suggesting potential for RNA delivery.
Area of Science:
- Cell biology
- Nanotechnology
- Biochemistry
Background:
- Microvesicles (MVs) and exosomes are cell-derived vesicles involved in intercellular communication.
- RNA molecules can be delivered to tissues via MVs and exosomes.
- Macrophages are known to internalize liposome-entrapped RNA molecules.
Purpose of the Study:
- To investigate the shedding of transfected RNA molecules from macrophages as MVs.
- To compare the secretion of chemically modified RNA (miR-143BPs) versus nonmodified RNA within MVs.
- To evaluate the in vivo delivery and distribution of MV-encapsulated RNA after intravenous injection.
Main Methods:
- Transfection of THP-1 cells (macrophages) with RNA molecules.
- Incubation in serum-free medium to induce MV shedding.
- Biochemical analyses including qRT-PCR and TSG101 expression.
- Immunoelectron microscopy for MV characterization.
- In vivo studies using xenografted nude mice to assess RNA distribution.
Main Results:
- Transfected RNA molecules were shed from THP-1 macrophages within MVs.
- Chemically modified miR-143BPs were secreted in MVs at higher levels than nonmodified RNA.
- Intravenous injection of transfected THP-1 macrophages led to increased miR-143 levels in mouse serum, tumors, and kidneys.
- These findings confirm in vitro and in vivo secretion of MV-encapsulated RNAs from macrophages.
Conclusions:
- Macrophages can secrete transfected RNA molecules encapsulated within microvesicles.
- Chemically modified RNAs show enhanced encapsulation and secretion within MVs.
- MV-encapsulated RNAs secreted by macrophages demonstrate potential for in vivo delivery to target tissues.
