Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes.
The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.
Sonawane
N D
ND
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA.
Szoka
Francis C
FC
Verkman
A S
AS
D000818:Animals; D002021:Buffers; D016466:CHO Cells; D002412:Cations; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D011992:Endosomes / Q000648:ultrastructure*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010873:Pinocytosis; D011073:Polyamines / Q000378:metabolism*; D011094:Polyethyleneimine; D011107:Polylysine / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011522:Protons; D012235:Rhodamines; D014162:Transfection
D002021:Buffers; D002412:Cations; D002712:Chlorides; D005456:Fluorescent Dyes; D011073:Polyamines; D011108:Polymers; D011522:Protons; D012235:Rhodamines; D011107:Polylysine; C005358:tetramethylrhodamine; D011094:Polyethyleneimine; D004247:DNA; D016650:Fluorescein-5-isothiocyanate