Chloride Accumulation and Swelling in Endosomes Enhances DNA Transfer by Polyamine-DNA Polyplexes*

By N. Sonawane, Francis C. Szoka Jr., A. Verkman. Journal of Biological Chemistry, 2003

982 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/0ddc3e0c29a12c074aee30705c6ad23065e7e6c3

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Citations in the corpus

Breaking down the barriers: siRNA delivery and endosome escape (2010, background): “Increased counter ion concentrations inside the endosome lead to osmotic swelling, endosomal membrane rupture, and the eventual leakage of the polyplex (cationic polymer complexed with nucleic acid) into the cytosol (Sonawane et al., 2003).”
Dendrimers in gene delivery. (2005, background): “This has been proposed as a general mechanism that facilitates escape from the endosome because of the accumulation of Cl and subsequent osmotic swelling of the endosome [98].”
Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling (2013, positive): “It has been hypothesized that the buffering capacity of nanoparticles can activate the proton pump that accentuates osmotic pressure inside an endosome, resulting in its swelling and subsequent endosomal escape of siRNA 7,8 .”
Exploring polyethylenimine‐mediated DNA transfection and the proton sponge hypothesis (2005, background): “Further, Sonawane and coworkers have recently shown that the concentration of endosomal chloride ions increases upon vesicle acidification and have observed the swelling/lysis of endosomes containing PEI polyplexes [22,29].”
Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives (2005, background): “Decelerated acidification, as well as elevated chloride accumulation and a 140% increase in the relative volume in PEI-containing endosomes, could be observed [30].”
PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles. (2004, background): “PEGylation and/or reaction to primary amines on the surface of bPEI polyplexes may impair the TMproton sponge∫ effect (Behr, 1994; Sonawane et al., 2003), a significant factor in the effectiveness of bPEI as a delivery vector.”
Nonviral Gene Delivery: Principle, Limitations, and Recent Progress (2009, neutral): “ethyleneimines (PEI) and partially degraded polyamidoamine dendrimers) absorb protons and slow down the acidification process that is essential for endosome–lysosome transition (7).”
Gene delivery by lipoplexes and polyplexes. (2010, background): “stable cylindrical bilayer, as indeed observed by Sternberg et al. (1994). Some studies have suggested that there is a simple and direct correlation between structure and transfection efficiency.”
Polyethylenimine, a cost-effective transfection reagent (2006, methodology): “Our data can be backed up with other studies using the unmodified PEI for successful transfection of various cell lines [1–3].”