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.