In vivo activation of PEGylated long circulating lipid nanoparticle to achieve efficient siRNA delivery and target gene knock down in solid tumors.

We developed a lipid nanoparticle formulation (LNPK15) to deliver siRNA to a tumor for target gene knock down. LNPK15 is highly PEGylated with 3.3% 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethylene glycol-2000) (PEG-DSPE) and shows a long duration: the half-lives of siRNA in LNPK15 were 15.2 and 27.0h in mice and monkeys, respectively. Although LNPK15 encapsulating KRAS-targeting siRNA (LNPK15/KRAS) had very weak KRAS gene knock down activity in MIA PaCa-2 cells in vitro, LNPK15/KRAS showed a strong anti-tumor efficacy in MIA PaCa-2 tumor xenograft mice after intravenous administration at 5mg/kg twice weekly. KRAS mRNA and protein knock down was observed in tumor tissue, suggesting on-target anti-tumor efficacy. In order to elucidate the in vitro-in vivo discrepancy, we performed ex vivo knock down assay using serum samples obtained after intravenous administration of LNPK15/KRAS to mice and monkeys. The collected samples were added to MIA PaCa-2 cells, and KRAS gene knock down was evaluated after a 24-h incubation period. The knock down efficacy was weak (≈20%) with serum samples at initial sampling point (2h), and it became much stronger (∼90%) with serum samples at later time points. Lipid composition of LNPK15 in the serum samples was also investigated. Among the five lipids incorporated in LNPK15, PEG-DSPE was degraded more rapidly than siRNA and the other lipids in both mice and monkeys. In vitro lipase treatment of LNPK15/KRAS also hydrolyzed PEG-DSPE and enhanced knock down activity. From these results, it was concluded that LNPK15 acquires increased knock down activity after undergoing PEG-DSPE hydrolysis in vivo, and that is the key mechanism to achieve both long circulation and potent knock down efficiency. We also proposed an in vitro assay system using lipase for quality control of LNP to ensure biological activity.

Design of a soluble transmembrane helix for measurements of water-membrane partitioning.

Use of model transmembrane helices and lipid bilayers is a tractable and straightforward approach to obtaining thermodynamic information on fundamental processes of membrane protein folding. The insertion of transmembrane helices from an aqueous phase into membranes, the initial step in the folding process, is especially difficult to investigate because of the insolubility of helices in the aqueous phase. We report here the design of a soluble transmembrane helix, (KR)(5)-AALALAA-AALWLAA-AALALAA-C(NBD)-NH(2) (NBD, 7-nitrobenz-2-oxa-1,3-diazole), consisting of a transmembrane region (AALALAA)(3), a central guest residue (W), and an N-terminal charged tag (KR)(5). Circular dichroism and fluorescence spectroscopy revealed that the peptide dissolved in water as a monomer with the guest residue exposed to the solvent. After the addition of large unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the peptide rapidly partitioned into the vesicles and assumed a transmembrane helix. The partitioning Gibbs free energy was estimated to be -34.2 kJ mol(-1) at 25 degrees C. The Trp-to-Gly substitution reduced the partitioning by approximately 1.6 kJ mol(-1). Thus, the transmembrane helix was found to be a useful template for thermodynamic measurements of the partitioning of amino acids from water to the hydrophobic core of membranes.