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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." />

Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes.

http://www.ncbi.nlm.nih.gov/pubmed/12944394

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.

Pubmed ID: 12944394 RIS Download

Mesh terms: Animals | Buffers | CHO Cells | Cations | Chlorides | Cricetinae | DNA | Endosomes | Fluorescein-5-isothiocyanate | Fluorescent Dyes | Hydrogen-Ion Concentration | Kinetics | Microscopy, Fluorescence | Pinocytosis | Polyamines | Polyethyleneimine | Polylysine | Polymers | Protons | Rhodamines | Transfection

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Associated grants

  • Agency: NIDDK NIH HHS, Id: DK35124
  • Agency: NIDDK NIH HHS, Id: DK46052
  • Agency: NIBIB NIH HHS, Id: EB00415
  • Agency: NIBIB NIH HHS, Id: EB03008
  • Agency: NEI NIH HHS, Id: EY13574
  • Agency: NHLBI NIH HHS, Id: HL59198
  • Agency: NHLBI NIH HHS, Id: HL73856

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