Polycation liposomes as a vector for potential intracellular delivery of microRNA
Hidenori Ando1, Ayaka Okamoto, Masafumi Yokota
1Department of Medical Biochemistry, University of Shizuoka Graduate School of Pharmaceutical Sciences, Shizuoka, Japan.
Background:
We previously developed a microRNA (miRNA) delivery system by using dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL), applied it to miR-92a delivery, and demonstrated its gene-silencing potential and effective anti-angiogenic effects. In the present study, we investigated the mechanism of intracellular delivery of cholesterol-grafted miR-92a (miR-92a-C) into cells.
Methods:
To investigate the intracellular distribution of miR-92a-C/TEPA-PCL complex, we used human umbilical vein endothelial cells and examined certain points after transfection: (i) the time-course of miR-92a-uptake into the cells; (ii) the endocytosis pathway induced by miR-92a-C/TEPA-PCL; (iii) the capability of miR-92a-C/TEPA-PCL to escape from the endosomes; and (iv) the release of miR-92a-C from TEPA-PCL in the cytoplasm.
Results:
Our data indicated that miR-92a-C formulated in TEPA-PCL accumulated in and was spread throughout the cytoplasm in a time-dependent manner, and was taken up into the cells by macropinosome-mediated endocytosis. In addition, the surface charge of miR-92a-C/TEPA-PCL was neutral at pH 7.4 and was charged positively at around pH 5.5, which is the inner pH of endosomes. When the late endosomes/lysosomes were stained with Lysotracker, miR-92a-C/TEPA-PCL efficiently escaped from the endosomes into the cytoplasm, possibly through the proton-sponge effect. Furthermore, miR-92a-C spread throughout whole cytoplasm and did not co-localize completely with TEPA-PCL, indicating that some of the miR-92a-C was present in free form in the cytoplasm.
Conclusions:
The results of the present study suggest that TEPA-PCL-based lipoplexes have an excellent potential to deliver microRNAs into the cytoplasm of cells and to induce RNA silencing action mediated by microRNAs and other small RNAs.
Insights
Cholesterol-grafted microRNA-92a (miR-92a-C) delivered via TEPA-PCL liposomes efficiently enters cells through macropinocytosis. The miR-92a-C is released into the cytoplasm, enabling potential RNA silencing actions.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Biology
Background:
- Previously developed tetraethylenepentamine-based polycation liposomes (TEPA-PCL) for microRNA (miRNA) delivery.
- Demonstrated gene-silencing and anti-angiogenic effects of miR-92a delivered by TEPA-PCL.
Purpose of the Study:
- Investigate the intracellular delivery mechanism of cholesterol-grafted miR-92a (miR-92a-C) using TEPA-PCL.
- Elucidate the uptake pathway, endosomal escape, and cytoplasmic release of miR-92a-C.
Main Methods:
- Utilized human umbilical vein endothelial cells for transfection studies.
- Analyzed time-course of miR-92a uptake, endocytosis pathway, endosomal escape, and cytoplasmic release.
- Examined the charge properties of miR-92a-C/TEPA-PCL at different pH levels.
Main Results:
- miR-92a-C/TEPA-PCL was taken up via macropinocytosis in a time-dependent manner.
- Lipoplexes exhibited pH-dependent charge changes, facilitating endosomal escape possibly via the proton-sponge effect.
- miR-92a-C was released into the cytoplasm, with some existing in a free form.
Conclusions:
- TEPA-PCL lipoplexes show significant potential for delivering miRNAs into the cell cytoplasm.
- This delivery system can effectively induce RNA silencing mediated by miRNAs and other small RNAs.
