Research Article
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
The Delivery of Small Interfering RNA to Hepatic Stellate Cells Using a Lipid Nanoparticle Composed of a Vitamin A-Scaffold Lipid-Like Material

https://doi.org/10.1016/j.xphs.2017.04.042Get rights and content

Abstract

Hepatic stellate cells (HSCs) are responsible for hepatic fibrosis and liver cirrhosis via their ability to produce extracellular matrices such as collagens and elastin. However, a strategy for delivering cargoes to HSCs has not been established yet. We herein report on attempts to deliver small interfering RNA (siRNA) to HSCs using several types of SS-cleavable proton-activated lipid-like materials (ssPalms) that contained myristic acid (ssPalmM) or hydrophobic vitamin A (ssPalmA) and E (ssPalmE) as hydrophobic scaffolds. We initially verified that hepatic fibrosis could induce the treatment with tetrachloromethane in terms of collagen fibrils and the expression of marker genes, type I collagen α-1, transforming growth factor β, heat shock protein 47, and α-smooth muscle actin. The siRNA silencing efficiency of the 3 LNPs was then compared using fibrosis-induced mice. Of the materials tested, LNPssPalmA showed the highest efficiency, with an effective (ED)50 of approximately 0.25 mg siRNA/kg. The LNPssPalmA showed a significant inhibitory effect on collagen production at a dose of 3.0 mg siRNA/kg with no evidence of any severe adverse effects. In conclusion, LNPssPalmA holds considerable potential for use in the treatment of HSCs-mediated diseases.

Introduction

It is well known that non-parenchymal cells in liver tissue, such as Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells (HSCs), play a key role in liver pathology in spite of their relatively small population in liver tissue (30%-35%).1, 2 HSCs are involved in the progression of fibrosis via the production of extracellular matrices including collagens.3 In healthy livers, HSCs store vitamin A and lipids for maintaining homeostasis.4 Although liver fibrosis can result in liver cirrhosis, and finally hepatocellular carcinomas, no effective treatment has yet been developed. Accordingly, the selective delivery of a cargo to HSCs would be a promising therapy for treating these diseases.

Unlike parenchyma cells (hepatocytes), no defined strategy is available for targeting HSCs using a drug delivery system. Hepatocyte-targeting neutral liposomes are preferentially taken up by interacting with apolipoprotein (Apo) E that is located on the surface of HSCs with the low-density lipoprotein receptor expressed by hepatocytes.5 Such interactions between lipoprotein receptors on hepatocytes and Apos are currently utilized for delivering therapeutics to hepatocytes.6, 7, 8 It should be noted that reports on targeting HSCs are sparse.

We recently developed a liposomal delivery platform for small interfering RNA (siRNA). The platform contained an SS-cleavable proton-activated lipid-like material (ssPalm) for delivering nucleic acids, including plasmid DNA and siRNA, to astrocytes,9 cancer cells,10 and hepatocytes.11 ssPalms consist of 2 tertiary amines that are linked via disulfide bonds in the hydrophilic region that are pH-sensitive and undergo auto-destabilization in response to acidification and reducing conditions in target cells, and a lipophilic moiety, such as myristic acid (ssPalmM), lipophilic vitamin A (ssPalmA) or E (ssPalmE). HSCs are one of most vitamin A-storing cells in the body.12 Based on this fact, we hypothesized that LNPs with a vitamin A scaffold had the potential for delivering therapeutics to HSCs. Accordingly, we evaluated a series of different lipid nanoparticles (LNPs) containing ssPalmA, ssPalmM, and ssPalmE for their silencing ability in HSCs.

Section snippets

Materials

siRNA (si-COL1A1, sense: gga auu cgg acu aga cau uTT, anti-sense: 5′-gga auu cgg acu aga cau uTT-3′, lower and upper cases denote RNA and DNA, respectively) was purchased from Hokkaido System Science (Sapporo, Japan). Primer sets were obtained from Sigma-Aldrich Japan (Ishikari, Japan). The primer sets used in this study are as follows: Col1a1, forward: TCA TCG ATG TGG CTC CCT TG, reverse: GGG TGG AGG GAG TTT ACA CG; Tgfb, forward: ATG CTA AAG AGG TCA CCC GC, reverse: ACT GTG TGA GAT GTC TTT

Preparation of ssPalm LNPs Containing Encapsulated siRNA

A 250 μL solution containing 75 μg of siRNA in 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer (pH 4.0) was gradually added to a 250 μL solution containing 36 μg protamine in HEPES buffer (pH 4.0). The ratio of siRNA and protamine was adjusted at a nitrogen/phosphate (N/P) ratio of 1.2. In another tube, 198 nmol of ssPalmX (X = M, A, or E), 198 nmol of di-oleoyl-sn-glycerophosphoethanolamine, and 264 nmol of cholesterol with 19.8 nmol of PEG-DMG (30/30/40 + 3 mol%)

Preparation of Liver Fibrosis Model

To assess the silencing effect of Palm LNPs in a fibrosis model, we produced a fibrosis model using CCl4. CCl4 in olive oil was continuously administered into ICR mice in the abdomen for 3 weeks (Fig. 1a). Liver tissues were excised 4 weeks after the first injection and collagen fibrils were stained with Sirius red (Fig. 1b), a characteristic stain that is specific for liver fibrosis.19 Red deposits, indicating collagen fibrils, were observed around the central veins. For further elucidation,

Discussion

This study focused on strategies for targeting HSCs using a series of original lipids developed in out laboratory, namely, an ssPalm with a hydrophobic vitamin instead of an alkyl chain. As mentioned above, HSCs are known to be one of the most efficient vitamin A-storage cells in the body and function to regulate the serum levels of vitamin A.27 Therefore, HSCs produce several vitamin A-binding proteins, both inside and on the outside of the cell. In previous reports, Niitsu et al. demonstrated

Conclusion

We developed a siRNA system that permits cargoes to be delivered to HSCs. The method is based on the ability of HSCs to store vitamin A, in which siRNA contained in LNPs that contain ssPalmA (LNPssPalmA) and vitamin A as a hydrophobic scaffold. In comparison to other LNPs, the LNPssPalmA exhibited delivered higher amounts of siRNA to HSCs. In conclusion, LNPssPalmA represents a promising device for use in the treatment of HSCs-mediated hepatological diseases.

Acknowledgments

The authors wish to thank to Dr. Milton S. Feather for modifying the manuscript. This study was supported partly by research grants (Research on Development of New Drugs, Health and Labour Sciences Research Grant, and Initiative for Accelerating Regulatory Science in Innovative Drug, Medical Device, and Regenerative Medicine) from the Japan Ministry of Health (H24B-14), Labour and Welfare, Research Program on Hepatitis from Japanese Agency for Medical Research and Development and Grant-in-Aid

References (38)

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    Jimenez Calvente et al developed a lipid-like nanoparticle to deliver COL1A1 siRNA and found that type I collagen expression was significantly inhibited in human HSC cell line LX-2 and carbon tetrachloride (CCl4)-induced fibrotic liver in mice without inducing innate immunity responses.30 Moreover, a vitamin A-coupled lipid-like nanoparticle was developed to carry COL1A1 siRNA, which significant inhibited collagen production without any severe adverse effects at a 3-mg siRNA/kg dose.31 Kaps et al also developed a cationic nanohydrogel particle to deliver COL1A1 siRNA for the treatment of CCl4-induced liver fibrosis in a mice model.32

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Conflicts of interest: Hokkaido University and the NOF Corporation hold patent-pending (PCT/JP2012/079160) on the ssPalm chemicals. H.A., K.T. and H.H. are the inventors of the patent.

The authors Naoyuki Toriyabe and Yu Sakurai contributed equally to this manuscript.

This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2017.04.042.

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