The study investigates the "proton sponge hypothesis," which posits that enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H⁺ buffering polyamines leads to increased endosomal Cl⁻ accumulation and osmotic swelling/lysis. The researchers measured Cl⁻ concentration, pH, and volume in endosomes after internalization of polyplexes composed of plasmid DNA and polylysine (POL), polyethylenimine (PEI), or polyamidoamine (PAM). They found that POL-polyplexes showed a 41% increase in Cl⁻ concentration and a 15% decrease in pH over 60 minutes, while PEI and PAM-polyplexes exhibited a 115% increase in Cl⁻ concentration and a slower pH decrease. The relative endosome volume increased by 20% for POL-polyplexes and 140% for PEI-polyplexes over 75 minutes. Endosome lysis was observed in PEI-containing endosomes after 45 minutes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The results support the proton sponge hypothesis, providing a rational basis for improving non-viral vectors for gene delivery.The study investigates the "proton sponge hypothesis," which posits that enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H⁺ buffering polyamines leads to increased endosomal Cl⁻ accumulation and osmotic swelling/lysis. The researchers measured Cl⁻ concentration, pH, and volume in endosomes after internalization of polyplexes composed of plasmid DNA and polylysine (POL), polyethylenimine (PEI), or polyamidoamine (PAM). They found that POL-polyplexes showed a 41% increase in Cl⁻ concentration and a 15% decrease in pH over 60 minutes, while PEI and PAM-polyplexes exhibited a 115% increase in Cl⁻ concentration and a slower pH decrease. The relative endosome volume increased by 20% for POL-polyplexes and 140% for PEI-polyplexes over 75 minutes. Endosome lysis was observed in PEI-containing endosomes after 45 minutes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The results support the proton sponge hypothesis, providing a rational basis for improving non-viral vectors for gene delivery.