title: Investigation of enzyme-sensitive lipid nanoparticles for delivery of siRNA to blood-brain barrier and glioma cells.
abstract: Clinical applications of siRNA for treating disorders in the central nervous system require development of systemic stable, safe, and effective delivery vehicles that are able to cross the impermeable blood-brain barrier (BBB). Engineering nanocarriers with low cellular interaction during systemic circulation, but with high uptake in targeted cells, is a great challenge and is further complicated by the BBB. As a first step in obtaining such a delivery system, this study aims at designing a lipid nanoparticle (LNP) able to efficiently encapsulate siRNA by a combination of titratable cationic lipids. The targeted delivery is obtained through the design of a two-stage system where the first step is conjugation of angiopep to the surface of the LNP for targeting the low-density lipoprotein receptor-related protein-1 expressed on the BBB. Second, the positively charged LNPs are masked with a negatively charged PEGylated (poly(ethylene glycol)) cleavable lipopeptide, which contains a recognition sequence for matrix metalloproteinases (MMPs), a class of enzymes often expressed in the tumor microenvironment and inflammatory BBB conditions. Proteolytic cleavage induces PEG release, including the release of four glutamic acid residues, providing a charge switch that triggers a shift of the LNP charge from weakly negative to positive, thus favoring cellular endocytosis and release of siRNA for high silencing efficiency. This work describes the development of this two-stage nanocarrier-system and evaluates the performance in brain endothelial and glioblastoma cells with respect to uptake and gene silencing efficiency. The ability of activation by MMP-triggered dePEGylation and charge shift is demonstrated to substantially increase the uptake and the silencing efficiency of the LNPs.
authors.affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.
authors.forename: Jonas
authors.initials: J
authors.lastname: Bruun
authors.affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.
authors.forename: Trine B
authors.initials: TB
authors.lastname: Larsen
authors.affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.
authors.forename: Rasmus I
authors.initials: RI
authors.lastname: Jølck
authors.affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Eliasen
authors.affiliation: H Lundbeck A/S, Biologics and Pharmaceutical Science, Valby, Denmark.
authors.forename: René
authors.initials: R
authors.lastname: Holm
authors.affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.
authors.forename: Torben
authors.initials: T
authors.lastname: Gjetting
authors.affiliation: Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, DTU Nanotech, Lyngby, Denmark.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Andresen
mesh_terms: D001812:Blood-Brain Barrier; D001921:Brain / Q000378:metabolism; D001932:Brain Neoplasms / Q000188:drug therapy / Q000473:pathology*; D002412:Cations; D045744:Cell Line, Tumor / Q000187:drug effects; D002784:Cholesterol / Q000737:chemistry; D004705:Endocytosis; D005434:Flow Cytometry; D020868:Gene Silencing; D005909:Glioblastoma / Q000188:drug therapy; D005910:Glioma / Q000188:drug therapy / Q000473:pathology*; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D055666:Lipopeptides / Q000737:chemistry; D020782:Matrix Metalloproteinases / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism
chemical_list: D002412:Cations; D008055:Lipids; D055666:Lipopeptides; D034741:RNA, Small Interfering; D011092:Polyethylene Glycols; D002784:Cholesterol; D020782:Matrix Metalloproteinases
keywords: BBB; angiopep; cleavable PEG-lipid; gene therapy; matrix metalloproteinase; nanocarrier