In vivo gene silencing in the liver: Comparison of siRNA-loaded non biodegradable vs. biodegradable nanohydrogel particles for antifibrotic therapy

Background:

Efficient in vivo transport of active siRNA to specific liver cells remains challenging. Compared to lipoplex formulations, cationic nanohydrogel particles (NHP) can serve as superior oligonucleotide carriers. We have designed a new generation of biodegradable NHP (bio-NHP) with improved biocompatibility properties that show promising in vitro and in vivo performance. Bio-NHP exhibit all NHP's positive characteristics like avoidance of carrier aggregation under physiological conditions and gene silencing in vitro by lacking any in vivo toxic effects even applied at high dosages up to corresponding 10 mg/kg siRNA.

Results:

Bio-NHP composed of block copolymers with acid-labile ketal linker in the inner core loaded with scrambled negative control siRNA (bio-NHP/scsiRNA) displayed a size around 40nm and did not show cytotoxicity for murine 3T3 fibroblasts, Raw or MHS macrophages, HepG2 human hepatoma cells or AML-12 murine benign hepatocytes up to concentrations of 400 nM scsiRNA. FITC-labelled bio-NHP were efficiently and dose-dependently taken up by 3T3 and RAW-cells, reaching 100% after 1h incubation as determined by FACS analysis. Additionally, FITC-bio-NHP exhibited a preferential uptake into CD14 human monocytes > CD19 B-cells > CD4/CD8 T-cells compared to NHP. Near infrared (NIR) Cw800-labeled bio-NHP loaded with Cy5-labeled antiprocollagen a1(I) siRNA (bio-NHP/col1a1) yielded a robust knockdown in 3T3 cells after 48h incubation. After intravenous injection, bio-NHP/col1a1 distributed preferentially to the liver (80%), as determined by in vivo and ex vivo NIR imaging, in normal and CCl4-fibrotic (6 weeks) mice. FACS analysis revealed a preferential colocalization of bio-NHP/col1a1 with primary myofibroblasts (α-SMA) > endothelial cells (CD31) > macrophages (CD45, F4/80) > hepatocytes (albumin). Bio-NHP/col1a1 generated an up to 80% in vivo procollagenIa1 knockdown in fibrotic mice, equivalent to the efficiency to NHP/col1a1. On the protein level, 2 treatments with bio-NHP/col1a1 significantly decreased liver collagen (Sirius red staining and hydroxyproline content), and also reduced CD68 positive macrophages.

Bio.-NHP loaded with negative control siRNA were eliminated by 50% after 72h and well tolerated up to 10 mg/kg negative control siRNA in fibrotic mice.

Conclusions:

siRNA loaded bio-NHP display attractive biocompatibility characteristics and confer efficient gene silencing properties in liver fibrogenic effector cells myofibroblast and will be further developed for sustained antifibrotic therapy.