Single cell kinetics of intracellular, nonviral, nucleic acid delivery vehicle acidification and trafficking.
Mechanistic understanding of the intracellular trafficking of nonviral nucleic acid delivery vehicles remains elusive. A live, single cell-based assay is described here that is used to investigate and quantitate the spatiotemporal, intracellular pH microenvironment of polymeric-based nucleic acid delivery vehicles. Polycations such as polyethylenimine (PEI), poly-l-lysine (PLL), beta-cyclodextrin-containing polymers lacking or possessing imidazole termini (CDP or CDP-imid), and cyclodextrin-grafted PEI (CD-PEI) are used to deliver an oligonucleotide containing a single fluorophore with two emission lines that can be employed to measure the pH. Delivery vehicles were also sterically stabilized by addition of poly(ethylene glycol) (PEG) and investigated. The intracellular trafficking data obtained via this new methodology show that vectors such as PEI and CDP-imid can buffer the endocytic vesicles while PLL and CDP do not. Additionally, the PEGylated vectors reveal the same buffering capacity as their unstabilized variants. Here, the live cell, spatiotemporal mapping of these behaviors is demonstrated and, when combined with cell uptake and luciferase expression data, shows that there is not a correlation between buffering capacity and gene expression.
Kulkarni
Rajan P
RP
Option in Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California 91125, USA.
Mishra
Swaroop
S
Fraser
Scott E
SE
Davis
Mark E
ME
D001483:Base Sequence; D001692:Biological Transport; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009696:Nucleic Acids / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D014677:Pharmaceutical Vehicles; D010957:Plasmids; D014162:Transfection
D009696:Nucleic Acids; D014677:Pharmaceutical Vehicles