Real time measurement of PEG shedding from lipid nanoparticles in serum via NMR spectroscopy.

Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems.

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.

The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.