The systemic administration of an anti-miRNA oligonucleotide encapsulated pH-sensitive liposome results in reduced level of hepatic microRNA-122 in mice
Graphical abstract
Introduction
MicroRNAs (miRNAs) are a class of small non-coding RNAs (~ 22 nt) that regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of target genes, triggering the degradation of messenger RNA (mRNA) or the inhibition of protein translation [1]. MicroRNA-122 (miR-122) is a conserved liver-specific miRNA that accounts for 70% of the total miRNA population [2] and plays important roles in liver physiology, such as lipid metabolism [3], [4], diseases in hepatic virus C (HCV) infections [5] and hepatocellular carcinoma (HCC) [6]. In addition, the expression of miR-122 in other organs such as heart is negligible [7]. Thus, miR-122 is an attractive and selective therapeutic target for the treatment of liver diseases [8].
A number of attempts have been made to induce the specific inhibition of endogenous miRNAs in vivo. Plasmid DNA vectors that express miRNA sponges, which contain multiple tandem miRNA binding sites, have been designed to competitively inhibit miRNA functions in mammalian cells [9]. It was recently reported that the intravenous administration of recombinant adeno associated virus (rAAV) vectors with miRNA tough decoys (TuDs) result in reduced levels of miR-122 and serum cholesterol [10]. Anti-microRNA oligonucleotides (anti-miRs) have been widely employed to inhibit miRNAs, including a variety of nucleoside modifications, such as 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and locked nucleic acid (LNA) was developed in an attempt to enhance binding affinity to target miRNAs with phosphorothioate (PS) linkages to improve nuclease resistance [11]. The systemic injection of these anti-miRs against miR-122 (anti-miR122) into mice resulted in a reduction in cholesterol levels [3], [4], [12], [13], [14]. Miravirsen, an LNA-modified anti-miR122, is currently in phase 2 clinical trials for the treatment of HCV [15].
However, large doses of anti-miR-122 are required to induce the phenotype when the free form of anti-miR-122 is injected due to renal excretion and poor tissue selectivity as well as cellular uptake [16], [17], [18]. To overcome these issues, systems that are capable of delivering nucleic acids to a targeted organ are desired [18]. Lipid based nanoparticles have been extensively investigated as vehicles for delivering siRNAs, miRNAs, as well as anti-miRs [19], [20], [21], [22], [23]. We developed a multifunctional envelope-type nano device (MEND), in which nucleic acids are encapsulated within a lipid envelope [24], [25]. We recently synthesized a pH-sensitive cationic lipid, referred to as YSK05 [26]. A MEND composed of YSK05 (YSK05-MEND) showed efficient pH-sensitive fusogenic properties and a higher gene knockdown ability than a commercially available transfection reagent, Lipofectamine 2000 (LFN2k) in HeLa cells [26]. The systemic administration of YSK05-MEND modified with PEG delivered siRNA to tumor tissue in renal cell carcinoma xenograft mice via the enhanced permeability and retention (EPR) effect and induced the knockdown of approximately 50% of the target gene at a dose of 3 mg/kg body weigh [27]. These findings indicate that YAK05-MEND has the potential for use in nucleic acid delivery both in vitro and in vivo.
In the present study, we report on an investigation of whether the YSK05-MEND could be applicable for use in an anti-miR-122 delivery system to murine liver. We first evaluated the physical properties of the YSK05-MEND encapsulating anti-miR-122 modified with 2′-OMe and PS linkages (AMO122) and its activity in murine hepatoma cells in comparison with LFN2k. For in vivo studies, we compared the activity of systemically administrated YSK05-MEND with free AMO in terms of distribution in the liver and kidney, and the effect of antagonism of target miR-122 on the increase in the expression level of genes that are regulated by miR-122 and the subsequent reduction in plasma cholesterol levels. The findings indicate that the YSK05-MEND has the potential for use in the efficient delivery of AMOs to murine liver.
Section snippets
Materials
Cholesterol (Chol) was purchased from AVANTI Polar Lipids (Alabaster, AL, USA). 1,2-Dimyristoyl-sn-glycerol-methoxypolyethyleneglycol 2000 ether (PEG-DMG) was purchased from NOF Corporation (Tokyo, Japan). YSK05 was synthesized as described previously [26]. Anti-miR against miR-122 (AMO122) (5′-AsCsAsAsAsCsAsCsCsAsUsUsGsUsCsAsCsAsCsUsCsCsA-3′) and Cy5-labeled AMO (Cy5-AMO) (5′-Cy5-AsCsGAUAAACGGUUGUCUACGsUsCsAs-3′) were purchased from Hokkaido System Science Co., Ltd. (Sapporo, Japan)
Characterization of the prepared AMO122-YSK05-MEND
The average diameter, pdi and zeta-potential of the AMO122-YSK05-MEND were 71 ± 2 nm, 0.20 ± 0.01 and 3.1 ± 0.5 mV, respectively. The characteristics of empty YSK05-MEND prepared by the same procedure without AMO122 were similar (72 ± 2 d. nm, pdi 0.24 ± 0.02, zeta-potential 7.3 ± 1.2 mV). The recovery and encapsulation efficiency of AMO122 determined with RiboGreen were 91 ± 6% and 99 ± 1%, respectively. The encapsulation of AMO122 was also confirmed by electrophoresis (Fig. S1a). The apparent pKa of
Discussion
MiRNAs have emerged as important post-transcriptional regulators of gene expression in biological processes [1]. Loss of function studies using miRNA gene knockout techniques are frequently utilized to explore the functions of miRNAs [11], [29]. However, the generation of genetic knockouts is difficult, complicating and time consuming. A widely employed approach in loss-of-function studies is to use chemically modified anti-miRs [3], [4], [5], [12], [13], [14], and LNA-modified anti-miR has
Conclusion
In the present study, we demonstrated the delivery of AMO to hepatocytes by the intravenous administration of AMOs encapsulated in a pH-sensitive YSK05-MEND. The use of the AMO122-YSK05-MEND resulted in the efficient enhancement of AldoA in vitro owing to its ability to escape from endosomes in comparison with LFN2k. Systemically administrated Cy5-AMO formulated in YSK05-MEND was delivered to the liver rather than the kidney, whereas free Cy5-AMO mainly accumulated in the kidney due to its low
Acknowledgment
This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Area “Nanomedicine Molecular Science” (No. 2306), a Grant-in-Aid for Scientific Research (grant No. 23249008) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan and the Special Education and Research Expenses of MEXT. The authors also with to thank De. Milton S. Feather for his helpful advice in writing the English manuscript.
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