title: Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.
abstract: Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.
authors.affiliation: Biomedical Engineering Department, Johns Hopkins University School of Medicine, USA.
authors.forename: Ron B
authors.initials: RB
authors.lastname: Shmueli
authors.forename: Nupura S
authors.initials: NS
authors.lastname: Bhise
authors.forename: Jordan J
authors.initials: JJ
authors.lastname: Green
mesh_terms: D000818:Animals; D004730:Endothelium, Vascular / Q000502:physiology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D051379:Mice; D053758:Nanoparticles / Q000032:analysis / Q000737:chemistry*; D011108:Polymers / Q000032:analysis / Q000737:chemistry*; D012171:Retinal Vessels / Q000166:cytology; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
chemical_list: D011108:Polymers; C507253:poly(beta-amino ester)