title: Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles: A mechanistic insight.
abstract: Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvβ3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvβ3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems.
authors.affiliation: Université Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, 1200 Brussels, Belgium.
authors.forename: Héloïse
authors.initials: H
authors.lastname: Ragelle
authors.affiliation: University of Copenhagen, Faculty of Health and Medical Sciences, Department of Pharmacy, Section for Biologics, Universitetsparken 2, 2100 Copenhagen O, Denmark.
authors.forename: Stefano
authors.initials: S
authors.lastname: Colombo
authors.affiliation: Université Catholique de Louvain, Molecules, Solids and Reactivity, Institute of Condensed Matter and Nanosciences, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Vincent
authors.initials: V
authors.lastname: Pourcelle
authors.affiliation: Université Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, 1200 Brussels, Belgium.
authors.forename: Kevin
authors.initials: K
authors.lastname: Vanvarenberg
authors.affiliation: Université Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, 1200 Brussels, Belgium.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Vandermeulen
authors.affiliation: Université Catholique de Louvain, Institut de Recherche Clinique et Expérimentale, Pole of Pharmacology and Therapeutics, 1200 Brussels, Belgium.
authors.forename: Caroline
authors.initials: C
authors.lastname: Bouzin
authors.affiliation: Université Catholique de Louvain, Molecules, Solids and Reactivity, Institute of Condensed Matter and Nanosciences, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Marchand-Brynaert
authors.affiliation: Université Catholique de Louvain, Institut de Recherche Clinique et Expérimentale, Pole of Pharmacology and Therapeutics, 1200 Brussels, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: Feron
authors.affiliation: University of Copenhagen, Faculty of Health and Medical Sciences, Department of Pharmacy, Section for Biologics, Universitetsparken 2, 2100 Copenhagen O, Denmark.
authors.forename: Camilla
authors.initials: C
authors.lastname: Foged
authors.affiliation: Université Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, 1200 Brussels, Belgium.
authors.forename: Véronique
authors.initials: V
authors.lastname: Préat
mesh_terms: D045744:Cell Line, Tumor; D048271:Chitosan / Q000008:administration & dosage*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D007097:Imines / Q000008:administration & dosage*; D016023:Integrins / Q000008:administration & dosage* / Q000235:genetics; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D053758:Nanoparticles / Q000008:administration & dosage*; D011092:Polyethylene Glycols / Q000008:administration & dosage*; D011095:Polyethylenes / Q000008:administration & dosage*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics
chemical_list: D007097:Imines; D016023:Integrins; D011095:Polyethylenes; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; C505405:poly(ethylene imine); D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D048271:Chitosan
keywords: Chitosan; Nanoparticles; RGD peptidomimetic; Stem-loop RT-qPCR; siRNA; α(v)β(3) Integrin