The Delivery of Small Interfering RNA to Hepatic Stellate Cells Using a Lipid Nanoparticle Composed of a Vitamin A-Scaffold Lipid-Like Material.

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 ß, 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.

New drug delivery system for liver sinusoidal endothelial cells for ischemia-reperfusion injury.

AIM: To investigate the cytoprotective effects in hepatic ischemia-reperfusion injury, we developed a new formulation of hyaluronic acid (HA) and sphingosine 1-phophate. METHODS: We divided Sprague-Dawley rats into 4 groups: control, HA, sphingosine 1-phosphate (S1P), and HA-S1P. After the administration of each agent, we subjected the rat livers to total ischemia followed by reperfusion. After reperfusion, we performed the following investigations: alanine aminotransferase (ALT), histological findings, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, and transmission electron microscopy (TEM). We also investigated the expression of proteins associated with apoptosis, hepatoprotection, and S1P accumulation. RESULTS: S1P accumulated in the HA-S1P group livers more than S1P group livers. Serum ALT levels, TUNEL-positive hepatocytes, and expression of cleaved caspase-3 expression, were significantly decreased in the HA-S1P group. TEM revealed that the liver sinusoidal endothelial cell (LSEC) lining was preserved in the HA-S1P group. Moreover, the HA-S1P group showed a greater increase in the HO-1 protein levels compared to the S1P group. CONCLUSION: Our results suggest that HA-S1P exhibits cytoprotective effects in the liver through the inhibition of LSEC apoptosis. HA-S1P is an effective agent for hepatic ischemia/reperfusion injury.

The transfection activity of R8-modified nanoparticles and siRNA condensation using pH sensitive stearylated-octahistidine.

Emerging evidence indicates that the efficiency of siRNA loading into an RNA-induced silencing complex (RISC) is a major factor in gene silencing at low doses. In particular, the release of siRNA from components delivered to the cytoplasm could be a first step for achieving maximum gene knockdown effect in siRNA delivery vector. To test this hypothesis, we used a stearylated-octahistidine (STR-H8) as a pH responsive polycation that facilitates the efficient release of siRNA into the cytoplasm, while a stearylated-octaarginine (STR-R8) was used as a conventional cationic polycation. As a fundamental structure, we used octaarginine (R8) and GALA, as a pH-sensitive fusogenic peptide, modified lipid envelope-type nanoparticles (R8/GALA-MEND(SUV)), as reported previously. When STR-H8/siRNA condensed complexes were loaded in the R8/GALA-MEND(SUV), the luciferase knockdown effect was significantly increased compared to STR-R8/siRNA condensed complexes in time and dose dependent manners. Quantification of the released siRNA from the condensed complexes demonstrated that only the STR-H8/siRNA released significant levels of siRNA at pH = 7.4, the pH of cytoplasmic, compared with STR-R8/siRNA condensed complexes. In addition, imaging studies indicated that STR-H8/siRNA facilitated siRNA release. Collectively, these data reveal the importance of the controlled release of siRNA to the cytoplasm.

Synthesis and evaluation of stearylated hyaluronic acid for the active delivery of liposomes to liver endothelial cells.

Hyaluronic acid (HA) is a naturally-occurring ligand that can be useful for targeting liver endothelial cells. We describe herein the development of a new HA-lipid conjugate for the efficient delivery of liposomes to liver endothelial cells. When free HA coated cationic liposomes were injected into mice, their accumulation in the liver was significantly decreased depending on the content of free HA, while accumulation in the lung was not significantly changed. When cationic liposomes modified with HA-stearylamine (HA-SA conjugate) were injected in mice, liver accumulation was increased depending on the amount of HA-SA conjugate and accumulation in the lung was drastically reduced, compared to non-modified liposomes. Confocal imaging analyses showed that HA-SA modified liposomes were accumulated to a greater extent along with blood vessels than non-modified liposomes, suggesitng that HA-SA modified liposomes are distributed in endothelial cells in the liver. Collectively, these findings indicate that an HA-SA conjugate is a useful material that can be used to modify liposomes and for delivering bioactive liposomal cargoes to liver endothelial cells.