title: Formulation of chitosan-DNA nanoparticles with poly(propyl acrylic acid) enhances gene expression.
abstract: Poly(propyl acrylic acid) (PPAA) is a polymer specifically designed to disrupt lipid bilayer membranes within a sharply defined pH range. The pH sensitivity can be used to enhance the release of endocytosed drugs into the cytoplasmic compartment of the cell. By incorporating this polymer in a polymeric gene carrier, chitosan, the release of plasmid DNA from the endosomal compartment was enhanced. In vitro transfection studies confirmed that the incorporation of PPAA into the chitosan-DNA nanoparticles enhanced gene expression in both HEK293 and HeLa cells compared to chitosan nanoparticles alone. The dose and time at which PPAA was incorporated during the complex formation affected the release of DNA and transfection efficiency. The optimal dose of PPAA incorporated into the chitosan nanoparticles was determined to be 10 microg, corresponding to a PPAA/DNA weight ratio of 1:1. At this dose, the ternary complexes are approx. 400 nm in size with a net negative surface charge of -17.4 mV. Intracellular trafficking studies confirmed the association of PPAA, DNA and chitosan at 24 h post-transfection and the subsequent release of DNA and PPAA from the chitosan at 48 h. The diffuse appearance of the majority of the DNA and the PPAA at later time points suggests that the PPAA triggered membrane disruption resulting in the release of DNA from the endosomal compartment. Finally, the lack of colocalization between PPAA and Lysotracker indicated that the PPAA-loaded nanoparticles were not trafficked through a lysosomal pathway. This study suggests the promising strategy of including PPAA in the formulation of polymer-DNA complexes for non-viral gene delivery.
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Tina
authors.initials: T
authors.lastname: Kiang
authors.forename: Corinne
authors.initials: C
authors.lastname: Bright
authors.forename: Charles Y
authors.initials: CY
authors.lastname: Cheung
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
authors.forename: Allan S
authors.initials: AS
authors.lastname: Hoffman
authors.forename: Kam W
authors.initials: KW
authors.lastname: Leong
mesh_terms: D000179:Acrylates / Q000737:chemistry*; D002460:Cell Line; D048271:Chitosan / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D018613:Microscopy, Confocal; D049329:Nanostructures; D010316:Particle Size; D011108:Polymers / Q000737:chemistry*
chemical_list: D000179:Acrylates; D011108:Polymers; C501628:poly(propyl acrylic acid); D004247:DNA; D048271:Chitosan